Unidirectional Notch signaling depends on continuous cleavage of Delta by Drosophila Kuzbanian-like

Unidirectional signaling from cells expressing Delta (Dl) to cells expressing Notch is a key feature of many developmental processes. The Drosophila ADAM metalloprotease Kuzbanian-like (Kul) has been shown to play a key role in promoting this asymmetry. Kul cleaves Dl efficiently both in cell culture and in flies, and has previously been shown not to be necessary for Notch processing during signaling. In the absence of Kul in the developing wing, the level of Dl in cells that normally receive the signal is elevated, and subsequent alterations in the directionality of Notch signaling lead to prominent phenotypic defects. Proteolytic cleavage of Dl by Kul represents a general mechanism for refining and maintaining the asymmetric distribution of Dl, in cases where transcriptional repression of Dl expression does not suffice to eliminate Dl protein (Sapir, 2005).

ADAM proteins have a characteristic domain signature, including a signal peptide followed by a pro-domain, a metalloprotease domain possessing a zinc-binding catalytic pocket, and a disintegrin domain. A cysteine-rich region is followed by a transmembrane domain and cytoplasmic tail. Sequence similarity searches have determined that the Drosophila genome harbors five ADAM-family metalloproteases. The full open reading frames of these metalloproteases were obtained from a combination of cDNA clones, reverse-transcription based on gene prediction, and 5' RACE in cases where the cDNAs did not cover the entire coding region (Sapir, 2005).

Among the five Drosophila metalloproteases, a single homolog was identified for TNF-alpha converting enzyme (TACE); two homologs were found for Meltrin-alpha, and two for ADAM10. Analysis of the ADAM family phylogenetic tree identified gene duplication events that took place most probably after the divergence of the ancestors of nematodes and insects. While Kuz shows a high degree of similarity to human ADAM10, another Drosophila protein, which was termed Kuzbanian-like (Kul), exhibits an even higher degree of similarity to ADAM10, especially in the disintegrin domain (which facilitates substrate recognition), as well as in the metalloprotease catalytic domain (Sapir, 2005).

In view of the ability of Kuz to cleave Dl, three other Drosophila ADAM proteins were examined for this activity. Dl was co-expressed with each of the ADAM proteins in Drosophila S2 cells, and the levels of Dl were monitored in the cells and in the medium, by probing with an antibody directed against the Dl extracellular domain. When Dl alone was expressed in the cells, basal cleavage by endogenous proteases was detected by an accumulation of the cleaved form of Dl in the medium. Co-expression of Kuz led to a marked elevation of Dl in the medium, concomitant with a reduction in the levels of the membrane-bound Dl in the cells. Two additional ADAM proteins, Kul and DTACE, exhibited a similar potency of cleaving and releasing Dl to the medium. By contrast, the expression of DMeltrin had no effect (Sapir, 2005).

Serrate (Ser) is a second ligand of Notch in Drosophila and is employed in more restricted biological settings. A similar profile of cleavage was also observed for Ser, which was cleaved by Kuz, Kul and DTACE, but only marginally by DMeltrin. Detection of efficient cleavage in S2 cells, which are grown in suspension, supports the notion of cell-autonomous cleavage. Thus, cleavage of Dl or Ser by ADAM proteins is likely to take place within the same cell, rather than between adjacent cells (Sapir, 2005).

To examine the biological roles of the ADAM proteins that can cleave Dl, it was necessary to compromise their activity in flies. Mutations or P-element insertions in DMeltrin, DTACE and kul are not currently available. Therefore double-stranded RNA (dsRNA) 'knock-down' constructs were generated for each of these genes, directed against a region of minimal similarity with the other family members (Sapir, 2005).

Activation of the Notch pathway is required many times during normal wing development. Especially notable is the role of Notch activation in restricting the width of the wing veins within a pro-vein territory. Utilizing the UAS-GAL4 system, dsRNA constructs of Drosophila ADAM metalloproteases were expressed in the wing. Expression of ds-DMeltrin or ds-DTACE did not lead to any detectable wing phenotypes, even when expressed under the regulation of the potent wing driver MS1096-GAL4. By contrast, induction of ds-kul expression by the same driver gave rise to distorted wings, and loss of the wing margin following induction by sd-GAL4. Expression of ds-kul by the weaker driver, sal-GAL4, resulted in two distinct adult wing phenotypes in the spalt-expression domain, which encompasses the region between veins L2-L4 -- formation of multiple wing hairs, and partial loss of veins. The first phenotype was found to be Notch-independent (Sapir, 2005).

The opposite phenotype with respect to vein loss, i.e. vein thickening, was observed when full-length Kul was overexpressed by sal-GAL4. These phenotypes suggest a functional link between Kul and Notch signaling in patterning the wing veins. Additional defects in the morphology of the wing were observed, and may stem from effects of Kul on other substrates independent of the Notch pathway, in accordance with the additional defects observed following ds-kul misexpression. Compromising the levels of Kuz by a ds-kuz construct resulted in vein thickening, representing a Notch loss-of function. This vein phenotype of ds-kuz is similar to the reported kuz loss-of-function wing phenotype, and is also consistent with observations in cell culture, which have demonstrated that ds-kuz RNA abolished the S2 step of Notch cleavage, while ds-kul RNA had no effect on Notch (Sapir, 2005).

In light of the high sequence similarity between Kul and Kuz, the specificity and activity of ds-kul was verified in cultured cells. When Kul was expressed in S2 cells, both a high molecular weight precursor form, and a cleaved, mature form were detected. Expression of ds-kul eliminated both forms of Kul, but did not affect Kuz protein levels, demonstrating the specificity of ds-kul (Sapir, 2005).

The activity of ds-kul was also examined in vivo. A broad distribution of kul transcripts was detected during all embryonic stages and in the wing imaginal discs. ds-kul expression in the wing reduced endogenous kul mRNA levels, illustrating the potency of ds-kul in vivo (Sapir, 2005).

The structural requirements for Kul function were characterized by examining in S2 cells protein maturation and the role of the different domains in promoting Dl cleavage. Basal cleavage of Dl was enhanced by co-expression of full-length Kul. By contrast, a Kul variant bearing an E-to-A substitution within the metalloprotease catalytic domain (E-A Kul), which abolishes catalytic activity, failed to cleave Dl. This demonstrates the need for an active protease domain in Kul. A similar mutation abolished the catalytic activity of Kuz (Sapir, 2005).

Interestingly, E-A Kul reduces Dl cleavage below the basal level, probably due to formation of a complex between E-A Kul and Dl that is refractive to cleavage by the endogenous proteases. Expression of this construct in the wing gives rise to broadened veins, again probably due to the sequestration of Dl. A similar inhibitory effect in cell culture was detected when expressing a Kul protein lacking the intracellular domain. This domain is important for correct trafficking and sorting of ADAM proteins. Finally, the role of pro-domain removal was analyzed by expressing a form of Kul in which cleavage was blocked by mutating two conserved amino acids at the putative pro-domain cleavage site. This variant full-length form of Kul again failed to carry out Dl cleavage, demonstrating the need of pro-domain removal in order to convert Kul to an active protease (Sapir, 2005).

In the pupal wing, activation of the Notch pathway by Dl contributes to refinement and restriction of the veins. Dl, expressed by the central pro-vein cells, activates Notch in the lateral pro-vein domain, forcing these cells to adopt an inter-vein fate, while the Dl-expressing cells themselves differentiate as veins. Reduction of Notch signaling causes lateral pro-vein cells to adopt a vein cell fate, leading to vein thickening in the adult wing. By contrast, ectopic expression of Dl by the inter-vein cells results in Notch activation in the central pro-vein cells, leading to vein loss (Sapir, 2005).

ds-kul expression gives rise to loss of veins, which is consistent with an effect on Notch signaling. To verify that this is indeed the case, the effects of compromising Kul levels in the pupal wing were monitored by a transcriptional reporter of Notch activation termed Gbe+Su(H)m8, and by following the expression of Dl. In a wild-type pupal wing 30 hours after pupariation, Dl protein is restricted to the future veins, while reporter expression is prominent in the lateral pro-vein cells and excluded from the vein cells. It is interesting to note that reporter expression is detected even several cell rows away from the vein cells, possibly resulting from the capacity of Dl-expressing cells to form far-reaching cellular extensions and protrusions (Sapir, 2005).

Expression of ds-kul by sal-GAL4 had a marked effect on the expression of Dl, as well as on the Notch reporter. Irregular expansion of Dl into the lateral pro-vein territory was observed, while in other parts of the wing Dl expression disappeared from the veins. Notch-reporter expression expanded into the vein cells. Elevation in Dl levels in the lateral pro-vein cells endowed them with the capacity to activate Notch signaling within the vein, demonstrating a role for Kul in maintaining unidirectional signaling by the Notch pathway. These patterns account for the loss of veins seen in the adult wing following expression of ds-kul. The irregular patterns observed in the pupal wing may represent snapshots of a dynamic sequence, which is initiated by expansion of Dl to the lateral pro-vein cells, followed by expansion of Notch activation and loss of Dl expression in the vein cells (Sapir, 2005).

The expansion of Dl-protein distribution in the pupal wing following ds-kul expression implies that Kul normally contributes to the restricted distribution of Dl in this tissue. To examine in more detail the capacity of Kul to cleave Dl in vivo, the larval wing imaginal disc was monitored. Dl protein, detected by an antibody recognizing the extracellular domain, is normally observed as a membrane-associated protein that is elevated in the vein and juxta-margin cells and excluded from the wing margin. Changes in Dl distribution were monitored in discs where Kul was overexpressed by the MS1096 driver. Dl membrane-associated staining in the wing pouch was diminished, and a residual punctate staining appeared, possibly reflecting endocytosis of secreted Dl. Normal Delta distribution was retained in the notum, where Kul was not overexpressed. Similarly, expression of Kul by sal-GAL4 eliminated Dl in the sal domain. To verify that Kul directly affects the cleavage of Dl, rather than Dl expression, clones were generated overexpressing Dl under the regulation of actin-GAL4, in the absence or presence of ectopic Kul. Indeed, the prominent appearance of Dl was completely abolished when Kul was co-expressed in the same clone (Sapir, 2005).

The capacity of Kul to cleave Ser has been demonstrated in S2 cells. To check if Kul can also cleave Ser in vivo, the distribution of Ser was analyzed following overexpression of Kul, using an antibody recognizing the Ser extracellular domain. Ser is normally expressed in the wing disc at this phase in a similar pattern to that of Dl, with a more pronounced appearance in the dorsal side of the pouch. The effects of Kul overexpression on Ser were distinct from the effect on Dl. Ser protein did not disappear, but instead displayed an altered, punctate localization within the cells. Kul overexpression also led to a uniform expansion of Ser expression, especially in the dorsal part of the pouch, where the expression of MS1096-GAL4 is more pronounced. It is not known in which compartment(s) Ser accumulates, nor the mechanism by which Kul overexpression leads to this sequestration (Sapir, 2005).

To test if Kul cleaves Dl cells autonomously, cell clones overexpressing Kul were generated. Dl staining was diminished only in the clone cells, implying a cell autonomous activity of Kul. There was no detectable change in Ser distribution within these small clones. The same cleavage assay carried out with Kuz overexpression revealed a different substrate specificity. Kuz cleaved both Dl and Ser within the overexpression clones. Like Kul, the activity of Kuz is cell autonomous, i.e. restricted to the cells overexpressing the protease (Sapir, 2005).

In the wing disc, Notch signaling plays a key role in defining and maintaining the margin, in two distinct signaling phases. Initially, the asymmetry between the dorsal and ventral compartments defines the margin and induces the expression of Wg by the future margin cells. The process is dictated by expression of Fringe only in the dorsal compartment, facilitating Notch signaling in the two cell rows comprising the border between the two compartments. The wing margin fate is subsequently maintained by complementary unidirectional signals between the margin and juxta-margin cells. In the margin, Notch signaling leads to the expression of Wg and Cut, the latter operating as a transcriptional repressor of Dl. In parallel, Wg activates expression of Dl and Serrate in the juxta-margin cells. High levels of Dl and Serrate prevent Notch activation in these cells. Thus a stable loop of two reinforcing signals is generated (Sapir, 2005).

It was interesting to examine the biological consequences of the response to Kul overexpression. Genetic removal of Dl or Ser alone was not sufficient to alleviate the dominant-negative effect of the remaining ligand on Notch signaling in the juxta-margin cells. However, in the case of Kul overexpression, both ligands were affected, i.e., Dl was efficiently cleaved and Ser was predominantly sequestered within the cells. An expansion was observed in the expression of the Notch-target genes wg and cut. In addition, the expression of Ser was broader. It is assumed that effective removal of Dl in conjunction with sequestration of Ser, give rise to alleviation of their dominant-negative effect on Notch signaling in the juxta-margin cells expressing these ligands. Thus, a residual level of Dl or Ser on the cell surface could trigger Notch signaling within these cells. Activation of Notch subsequently leads to ectopic production of Wg, which in turn spreads to neighboring cells to trigger ectopic expression of Ser (Sapir, 2005).

Does endogenous Kul in the wing disc have a role in maintaining the asymmetric distribution of Dl? Expression of ds-kul by the potent sd-GAL4 driver gives rise to loss of Cut and Wg expression in the margin, and a reduction in the size of the wing pouch. The adult wings that develop are significantly reduced in size and show no indication of veins, and only rudimentary margin bristles in very restricted domains. These results demonstrate that Kul is essential for maintaining the spatial balance of Notch signaling in the wing margin (Sapir, 2005).

Induction of ds-kul by the sal-GAL4 driver does not affect the adult wing margin and results only in a reduction in Wg levels in the wing margin, without a pronounced effect on Cut levels. In view of the role Kul plays in Dl cleavage, it was of interest to test whether the changes in Notch target-gene expression in the wing margin result from elevation in Dl levels within the margin cells. Indeed, higher levels of Dl could be detected in the margin within the sal domain where ds-kul is expressed. By contrast, no effects of ds-kul on the distribution of Ser were observed (Sapir, 2005).

How does Kul activity impinge on the distribution of Dl? Overexpression of Kul in the wing disc results in a dramatic diminution of the levels of Dl. This effect is cell autonomous, i.e., Kul can only eliminate Dl within the cells in which Kul is expressed. It is not known if cleavage takes place once both proteins are localized to the cell surface, or if removal of Dl occurs during trafficking to the cell surface. Since no accumulation of Dl was observed within the cells following Kul overexpression, the first possibility is favored. Kul activity appears to be constitutive (see below), implying that there is no preferential cleavage of Dl by Kul in the receiving cells. Rather, the final outcome is likely to result from the activity of Kul in both cell types. In the receiving cells, where the levels of Dl are low, the proteolytic activity of Kul effectively eliminates the Dl protein. By contrast, in the sending cells expressing high levels of Dl, while Kul may cleave some of the ligand, sufficient levels of Dl remain to allow efficient signaling (Sapir, 2005).

Disruption of Notch unidirectional signaling following removal of Kul highlights the necessity of continuously removing the Dl protein, in order to generate a setting in which it would be hard for the Dl protein to accumulate. Transcriptional repression of Dl expression is not sufficient. For example, in the wing margin, activation of the Notch pathway specifically leads to the induction of E(spl) and Cut, which are transcriptional repressors of Dl expression. Yet, in the absence of Kul, some Dl protein is produced by the margin cells. Similarly, in the pupal wing, activation of Notch in the lateral pro-vein cells induces E(spl) expression. Nevertheless, Dl is produced by these cells when Kul is eliminated. These observations underscore an inherent difficulty in shutting down Dl transcription efficiently. They also imply that even residual levels of Dl have detrimental biological consequences. The constitutive cleavage of Dl by Kul is therefore a crucial safeguard, continuously removing low levels of Dl that have escaped transcriptional repression (Sapir, 2005).

The biological role of Kul was demonstrated in this work in two stages in which Notch signaling refines a pre-existing asymmetry between adjacent cells: the wing margin and the wing veins. In other instances, Notch signaling actually generates the asymmetry between cells. Notch defines the correct number and spacing of differentiated cells within a field of equipotent cells, e.g. in the embryonic neuroectoderm or among pupal sensory organs. In these cases, it is thought that stochastic fluctuations in the levels of Dl, coupled to mechanisms that amplify these changes, lead to differentiation of some cells and concomitant repression of differentiation in the neighboring cells. Kul does not seem to impinge on these process. No effects on the number and organization of neuroblasts were observed following induction of ds-kul by broad maternal and early zygotic drivers. Another avenue of Notch signaling is triggered by asymmetric cell divisions in the sensory neuron precursors. Again, induction of ds-kul by neu-GAL4 does not give rise to any Notch-related phenotypes in the sensory bristles. It is therefore concluded that the activity of Kul appears to be essential for Notch signaling specifically in cases where a pre-existing spatial asymmetry is used to guide the directionality of Notch signaling (Sapir, 2005).

In view of the central role of Kul in Notch signaling, it was important to examine the different junctions in which Kul activity may be regulated. At the transcriptional level, Kul appears to be broadly expressed, in embryos and in imaginal discs. This broad expression is also reflected in the Notch-independent multiple-wing hair phenotype that is observed in all wing cells where ds-kul is induced. It is still possible that the basal level of Kul expression may be elevated in cells where Notch signaling takes place, to reduce the levels of Dl in these cells more efficiently (Sapir, 2005).

At the post-transcriptional level, however, there are several steps in the generation of an active Kul protein, which could be regulated. The protein must be correctly targeted to the plasma membrane, a process that may rely on the cytoplasmic domain of Kul and its interaction with the intracellular trafficking machinery. The precursor form of Kul undergoes processing by Furins, to remove the pro-domain. In the absence of this processing, Kul cannot cleave Dl. Finally, association of Kul with its substrates is mediated by the disintegrin domain, and possibly also by additional proteins that could bias this interaction (Sapir, 2005).

In spite of the sequential processes necessary for the formation of a mature, active Kul protein, there is no evidence that any of these steps is regulated in time or space. The data so far support the notion of a constitutive maturation and processing of Kul. In every cell where Kul was misexpressed, an outcome was observed, as monitored by removal of Dl. In the wing, removal of Kul activity also gives rise to additional phenotypes that are not related to Notch, e.g., the appearance of multiple wing hairs. This phenotype was observed in all cells where ds-kul was expressed, again supporting the notion that Kul is normally expressed and activated uniformly (Sapir, 2005).

In conclusion, while Kul may be broadly active, it enhances and maintains the asymmetrical activation of Notch, by relying on the initial differences in the levels of Dl. Kul effectively removes the ligand from the cells expressing Dl at low levels, while retaining sufficient levels of Dl in the cells that will activate Notch. Thus, a uniform activity of Kul can amplify a bias in the levels of Dl expression, and leads to a strict unidirectional activation of Notch, a process that is central to patterning the organism at multiple stages of development (Sapir, 2005).

Drosophila follicle cells are patterned by multiple levels of Notch signaling and antagonism between the Notch and JAK/STAT pathways; Kuzbanian-like reduces the level of Delta signaling between follicle cells

The specification of polar, main-body and stalk follicle cells in the germarium of the Drosophila ovary plays a key role in the formation of the egg chamber and polarisation of its anterior-posterior axis. High levels of Notch pathway activation, resulting from a germline Delta ligand signal, induce polar cells. This study shows that low Notch activation levels, originating from Delta expressed in the polar follicle cells, are required for stalk formation. The metalloprotease Kuzbanian-like, which cleaves and inactivates Delta, reduces the level of Delta signaling between follicle cells, thereby limiting the size of the stalk. Notch activation is required in a continuous fashion to maintain the polar and stalk cell fates. Mutual antagonism between the Notch and JAK/STAT signaling pathways provides a crucial facet of follicle cell patterning. Notch signaling in polar and main-body follicle cells inhibits JAK/STAT signaling by preventing STAT nuclear translocation, thereby restricting the influence of this pathway to stalk cells. Conversely, signaling by JAK/STAT reduces Notch signaling in the stalk. Thus, variations in the levels of Notch pathway activation, coupled with a continuous balance between the Notch and JAK/STAT pathways, specify the identity of the different follicle cell types and help establish the polarity of the egg chamber (Assa-Kunik, 2007).

Stalk formation between adjacent egg chambers is induced by directional signaling from the anterior polar cells of the older (posterior) egg chamber. Signaling via the JAK/STAT pathway provides an essential component of this process, but various indications have suggested a role for the Notch pathway as well. To verify the requirement for Notch signaling in the induction of stalk cells, follicle cell clones were generated that are mutant for Dl, the primary Notch ligand during oogenesis. Despite the proper specification of polar cells, egg chambers containing Dl follicle cell clones often failed to form a stalk on their anterior side, and as a result fused to the neighboring egg chamber. Such clones always encompassed follicle cells at the anterior portion of the egg chamber, indicating that Dl produced by anterior follicle cells is necessary to form an anterior stalk. However, the stalk positioned on the posterior side of these egg chambers was normal, even when the Dl clone surrounded the entire germline cyst. This is in keeping with the observation that posterior follicle cells do not contribute to stalk formation (Assa-Kunik, 2007).

In order to determine which cells of the anterior follicle cell population provide the signal for stalk formation, small anterior Dl-mutant follicle cell clones were analyzed. In all cases where Dl-mutant clones led to loss of the stalk, the anterior polar cells were included in the mutant clone, suggesting that these cells are the source of Dl signaling. A few instances were observed in which an anterior stalk formed even though both polar cells were mutant for Dl. Since the polar cell population defined by expression of Fng is initially larger, and is reduced to two cells by programmed cell death, this most probably resulted from the presence of wild-type Dl-expressing polar cells that provided the signal prior to their apoptosis. No phenotype was observed when the stalk cells themselves were mutant for Dl, indicating that Dl production by the stalk cells is not required for stalk specification (Assa-Kunik, 2007).

These results indicate that Notch signaling is required for at least two processes of follicle cell patterning during early oogenesis: specification of polar cells induced by Dl from the germ line and induction of stalk by Dl provided by anterior polar cells. How are these two signals distinguished, and what is the temporal relationship between them (Assa-Kunik, 2007)?

The universal Notch transcriptional reporter Gbe+Su(H)m8-lacZ was to follow the activation profile of Notch signaling throughout oogenesis. During stages 2-3 of oogenesis, variations were observed in the strength of Notch pathway activation within different anterior follicle cell types. Activation of Notch was observed in the polar cells, but no activation could be detected at this resolution in the stalk cells. These observations indicate that the level of Notch activation in the stalk cells is significantly lower than in the polar cells. Utilization of a second Notch reporter (m7-lacZ) identified essentially the same pattern. However, as this reporter appears to be more sensitive than Gbe+Su(H)m8-lacZ, low levels of Notch activation in the stalk cells at early stages could also be observed (Assa-Kunik, 2007).

Expression of Fng specifically in the future polar cells, provides a possible basis for the enhanced magnitude of Notch signaling in these cells. Polar cells are also part of the follicle cell population adjacent to the germline nurse cell complex, in which overall levels of Dl protein appear relatively high. However, the fraction of Dl localized to the nurse-cell membranes is difficult to quantify, preventing attribution with confidence the differences in signaling levels during early oogenesis to this parameter (Assa-Kunik, 2007).

To define the temporal sequence of polar and stalk cell induction, the expression of specific markers was followed for each cell type. Polar and stalk cell markers are first detected in stage 1 egg chambers (region 3 of the germarium). Markers of both cell types could be detected simultaneously in some egg chambers, where they were aligned as broad adjacent bands, with the polar cell marker always positioned towards the posterior. All other egg chambers at this stage displayed expression of the polar cell marker alone. These observations imply that polar cells are induced first, and, in agreement with the genetic evidence, are properly positioned to signal and induce stalk cell formation at the anterior end of the egg chamber (Assa-Kunik, 2007).

Taken together, these data suggest that distinctions in both the strength of signaling via the Notch pathway and the temporal sequence of pathway activation contribute to distinct cell-fate outcomes within the population of anterior follicle cells during early Drosophila oogenesis (Assa-Kunik, 2007).

It has been shown that the metalloprotease Kuzbanian-like (Kul) cleaves Dl in a cell-autonomous manner, leading to its downregulation. Modulation of Kul levels therefore provides a sensitive tool for manipulating Dl signaling activity in vivo. Attempts were made to determine whether Kul functions within follicle cells during early oogenesis. The expression pattern of Kul during oogenesis was monitored by fluorescent RNA in situ hybridization. Whereas Kul RNA was not detected in the germ line, prominent expression was observed in follicle cells, up to stage 3 (Assa-Kunik, 2007).

Kul levels can be effectively reduced by expression of a specific UAS-dsRNA construct. Since expression of Kul dsRNA by various GAL4 drivers resulted in lethality, expression of this construct was restricted to adult stages through the use of a temperature-sensitive GAL80 inhibitor system. This approach was used throughout the study to enable expression of various UAS-based transgenes during oogenesis. The GAL80ts system was used in conjunction with the neur-GAL4 driver (A101-GAL4) to specifically express Kul dsRNA in polar cells, and assess the effect of Kul on Notch signaling in early follicle cells. Notch transcriptional reporter activity was examined in these egg chambers, and the position and intensity of staining compared with wild-type egg chambers that were processed under identical conditions. Following expression of dskul in polar cells, Notch reporter levels were significantly elevated, both in the germarium and in stage 1-3 egg chambers. These observations indicate that Kul acts as an attenuator of Dl signaling in early-stage follicle cells. Interference with Kul function in this fashion thus provides a means to address the significance of follicle cell Dl levels for proper stalk cell induction. Indeed, expression of dskul in the polar cells led to a significant increase in stalk-cell number, from an average of 7.0 to 10.3 cells per stalk (Assa-Kunik, 2007).

These results indicate that the size of the stalk is highly sensitive to the amount of Dl signaling between follicle cells. This is in agreement with previous experiments, in which the size of the stalk was dramatically increased following a mild hyperactivation of Notch. Consistent with these data, ovaries from heterozygous Dl females have a reduced number of stalk cells, underscoring the sensitivity of the system to levels of Dl signaling (Assa-Kunik, 2007).

To determine whether stalk cells remain sensitive to Notch pathway signaling following their differentiation, dskul was expressed in the stalk cells themselves, using the 24B-GAL4 stalk cell-specific driver, and an increase was observed in the number of stalk cells to an average of 9.0. Kul thus attenuates Dl levels even after the stalk is formed, implying that stalk-cell number is regulated by Dl signaling from both polar cells and the stalk cells themselves. In a converse experiment, Notch signaling was reduced or eliminated from the stalk cells. Expression of dsNotch, or of a dominant-negative Notch construct, by the 24B-GAL4 stalk cell-specific driver led to the disappearance of the stalk marker Big brain (Bib). Thus, persistent, low level activation of Notch is required to maintain stalk cell fate. The low levels of Dl employed for this purpose are presented initially at the polar cell-stalk cell boundary, but as the stalk becomes elongated they might be displayed by neighboring stalk cells (Assa-Kunik, 2007).

Dl is required for establishment and maintenance of the stalk cell fate. The sensitivity of stalk size to the levels of Dl provided by the stalk cells themselves suggests that Dl also affects stalk cell proliferation or survival. To examine this possibility, the anti-apoptotic protein p35 was expressed in both polar and stalk cells using the 109-53-GAL4 driver. A greater abundance was observed of cells not properly arranged into a one-cell-wide stalk. This suggests that excess stalk cells are normally eliminated by apoptosis, and would support a model in which Dl is required for stalk cell survival, as well as stalk differentiation (Assa-Kunik, 2007).

The above observations suggest that different levels of Notch signaling determine the final fate of cells from within the polar/stalk precursor population - a strong germline signal induces the polar cell fate, whereas a weaker follicle cell signal induces the stalk. As an additional test of this model, the effects were examined of strongly elevating the Notch follicle cell signal, by overexpression of Dl specifically in polar cells. Overexpression of Dl using polar cell-specific GAL4 drivers had dramatic effects on anterior follicle cell fate and tissue morphology. Significantly, this alteration in Notch signaling resulted in an excess of polar cells. Supernumerary polar cells formed primarily at the expense of stalk cells, as evidenced by their expression of both polar and stalk cell markers, and as fusions between adjacent egg chambers. Some of the excess polar cells expressed the main-body follicle cell marker Eya, suggesting that the elevated Dl signal was capable of recruiting polar cells from this neighboring population as well. Furthermore, overexpression of Dl within the stalk cells themselves, using the 24B-GAL4 driver, induced the expression of a polar cell marker within the stalk (Assa-Kunik, 2007).

The JAK/STAT ligand Upd is expressed in polar cells, and like Dl is required for induction of the stalk. The binding of Upd to its receptor, Domeless, activates the JAK kinase Hopscotch, which then phosphorylates STAT (Stat92E) to induce its translocation into the nucleus, where it regulates transcription. The observed shift from stalk to polar cell fate upon overexpression of Dl implies that Notch activation has the capacity to antagonize JAK/STAT signaling. To explore this issue further, the Notch m7-lacZ and the STAT92E-GFP transcriptional reporters were used to simultaneously monitor Notch and JAK/STAT signaling in the ovary. Two distinct distributions of transcriptional activation wee observed. During early stages of oogenesis, Upd signaling from the polar cells is capable of inducing strong STAT activation in stalk cells, but fails to elicit activation in either the polar cells themselves, or in the neighboring main-body follicle cells. At later stages, however, follicle cell populations, including main-body and border cells, exhibited concomitant Notch and STAT activation. This analysis highlights a continuous requirement for both the Notch and JAK/STAT signaling pathways during follicle cell differentiation, throughout oogenesis. As predicted, Notch signaling can antagonize STAT activation in follicle cells, but this capacity is spatially and temporally restricted (Assa-Kunik, 2007).

The antagonistic effect of Notch signaling in early egg chambers was further pursued by following nuclear localization of STAT as an assay for JAK/STAT pathway activity. Nuclear STAT staining was pronounced throughout the stalk separating the germarium from the polar cells of the adjacent, posterior egg chamber in wild-type ovaries. Consistent with the STAT92E-GFP reporter pattern, the anterior polar cells did not exhibit nuclear localization of STAT, indicating that although they produce the Upd ligand, they themselves are refractory to this signal. STAT also remained cytoplasmic in the main-body follicle cells adjacent to the polar cells (Assa-Kunik, 2007).

When Upd was overexpressed using a polar cell-specific driver, the anterior range of nuclear STAT localization was significantly increased. Consistent with this enhanced activation of JAK/STAT signaling, longer stalk-like structures were observed. In spite of the higher levels of Upd, nuclear STAT was still only seen in cells anterior to the source, including the future stalk and posterior polar cells of the adjacent younger egg chamber. By contrast, JAK/STAT signaling in the anterior polar cells themselves, and in the neighboring main-body follicle cells, was not activated (Assa-Kunik, 2007).

In light of the suggestion of an antagonistic relationship between Notch and JAK/STAT signaling, one possible explanation for failure of the polar and main-body follicle cells to respond to Upd is the higher level of Notch activation in these cells. To test this hypothesis, Notch-mutant clones were generated in the main-body follicle cells, and the nuclear localization of STAT was monitored. Elimination of Notch in these cells led to nuclear accumulation of STAT in mutant cells situated within four cell-diameters of the polar cells. No nuclear localization was detected in Notch-mutant cells situated further away, presumably owing to restricted diffusion of Upd from the polar cells (Assa-Kunik, 2007).

These results indicate that moderate to high levels of Notch activation inhibit JAK/STAT signaling, and that this inhibition acts before the nuclear translocation of activated STAT. Furthermore, the results demonstrate that correct specification of the polar, main-body and stalk follicle cells depends on crosstalk between distinct levels of Notch activity and the JAK/STAT pathway. High Notch activation induces polar cell fate, including expression of Upd, and antagonizes JAK/STAT signaling. Intermediate levels of Notch activation in the main-body follicle cells antagonize JAK/STAT signaling, without inducing expression of Upd. Finally, low levels of Notch activation synergize with Upd signaling to induce stalk cell fate and to regulate the size of the stalk (Assa-Kunik, 2007).

Maintaining the moderate level of Notch signaling that is induced by Dl expressed in the follicle cells, is essential for producing a stalk with the correct cell number, and this is achieved at least in part by the activity of Kul in the signal-sending cells. The possibility that Notch signaling is also attenuated in the signal-receiving cells by the activity of JAK/STAT was examined by monitoring oogenesis in hopscotch (hop) hypomorphs, in which JAK/STAT signaling is compromised. Stalks formed at early stages of oogenesis in hopmv1/GA32 females, and the oocyte moved to the posterior of the egg chamber as in wild type. However, stalk cells failed to intercalate, and the stalk consisted of two rows of cells linked by adherens junctions. At later stages, the stalk collapsed and, as was observed for strong hop alleles, the stalk cells reverted to the polar cell fate. These cells now clustered at the anterior corners of the older cyst, whilst remaining in contact with the oocyte of the younger egg chamber (Assa-Kunik, 2007).

The conversion of stalk cells to polar cells when the level of JAK/STAT signaling was compromised suggests that Notch signaling in the stalk cells is normally attenuated by the JAK/STAT pathway. When this inhibition is relieved in hop hypomorphs, the increase in the level of Notch signaling leads to their conversion to polar cells. Since the entire polar/stalk precursor cell population expresses Fng, even activation by the lower levels of Dl produced by these cells may be sufficient to give rise to polar cells, in the absence of repression by JAK/STAT (Assa-Kunik, 2007).

C. elegans Kuzbanian homologs

The ectodomain of LIN-12/Notch proteins is cleaved and shed upon ligand binding. In Caenorhabditis elegans, genetic evidence has implicated SUP-17, the ortholog of Drosophila Kuzbanian and mammalian ADAM10, as the protease that mediates this event. In mammals, however, biochemical evidence has implicated TACE, a different ADAM protein. This study investigated potential functional redundancy of sup-17 and the C. elegans ortholog of TACE, adm-4, by exploring their roles in cell fate decisions mediated by lin-12/Notch genes. It was found that reduced adm-4 activity, like reduced sup-17 activity, suppresses an allele of glp-1 that encodes a constitutively active receptor. Furthermore, concomitant reduction of adm-4 and sup-17 activity causes the production of two anchor cells in the hermaphrodite gonad, instead of one—a phenotype associated with loss of lin-12 activity. Concomitant reduction of both sup-17 and adm-4 activity in hermaphrodites results in highly penetrant synthetic sterility, which appears to reflect a defect in the spermatheca. Expression of a truncated form of LIN-12 that mimics the product of ectodomain shedding rescues this fertility defect, suggesting that sup-17 and adm-4 may mediate ectodomain shedding of LIN-12 and/or GLP-1. The results are consistent with the possibility that sup-17 and adm-4 are functionally redundant for at least a subset of LIN-12/Notch-mediated decisions in C. elegans (Jarriault, 2005: full text of article).

Vertebrate Kuzbanian homologs

A mouse protein (MKUZ) is 45% identical in primary sequence with Drosophila Kuz and 95% identical wth the bovine protein. Sequence similarity between MKUZ and Drosophila Kuz extends over the whole coding region, except that MKUZ, like other vertebrate KUZ homologs, has a much shorter intracellular domain. The intracellular domain of MKUZ contains a stretch of 9 amino acid residues that are absolutely conserved with DKuz. To determine the functional importance of this sequence similarity, mutations were introduced into KUZDN in these conserved residues. These mutations dramatically reduce KUZDN activity (Pan, 1997).

LIN-12/NOTCH proteins mediate cell-cell interactions that specify cell fates. Previous work has suggested that sup-17 facilitates lin-12 signaling in Caenorhabditis elegans. sup-17 encodes a member of the ADAM family of metalloproteases. SUP-17 is highly similar to Drosophila Kuzbanian, which functions in Drosophila neurogenesis, and the vertebrate ADAM10 protein. Bovine ADAM10 has been shown ot biochemically cleave myelin basic protein and proTNF alpha. The extracellular domain of LIN-12 appears to be necessary for sup-17 to facilitate lin-12 signaling; sup-17 does not act downstream of lin-12. Cell ablation experiments show that sup-17 can act cell autonomously to facilitate lin-12 activity. An important challenge for the future will be to elucidate the function of the processing of LIN-12/Notch extracellular domain with respect to ligand dependent activation and signal transduction (Wen, 1997).

The Notch receptor, which is involved in numerous cell fate decisions in both invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). Proteolytic processing of p300 is an essential step in the formation of the biologically active receptor because only the cleaved fragments are present at the cell surface. These results confirm and extend recent reports indicating that the Notch receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. Constitutive processing of murine Notch1 involves a furin-like convertase. This enzyme belongs to a family of subtilisin-like, calcium-dependent convertases that process proproteins in the constitutive secretory pathway, and furins belong to a different family of proteases than do ADAMs. The calcium ionophore A23187 and the alpha1-antitrypsin variant, alpha 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed. In vitro digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651-1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolishes processing of the Notch1 receptor (Logeat, 1998).

Recently it has been suggested that the disintegrin metalloprotease Kuzbanian is required for the constitutive cleavage of Notch. Overexpression of a dominant negative mutant of Drosophila Kuz (KUZ-DN) lacking the pro- and metallo-protease domains results in no detectable p120 Notch, neither in transfected S2 cells nor in Drosophila imaginal discs. No processing of Notch can be observed in kuz null Drosophila embryos. In the current experiments, the expression of KUZ-DN does not affect mouse Notch1 processing, irrespective of the amount of this dominant negative molecule introduced into cells. In any case, further work will be necessary to clarify these apparent discrepancies between the Drosophila and mammalian results and determine whether these discrepancies are because of differences in the experimental systems used, for example, between Drosophila and mammals. Because Kuz is required for the lateral inhibition process during Drosophila neurogenesis and its target seems to be the extracellular region of Notch, it is possible that Kuz is not involved in the constitutive maturation of the receptor but in a subsequent processing step that would follow interaction with the ligand. This might explain the recently published observation that a new band migrating slightly faster than p120 is induced by contact between cells expressing Notch1 and cells expressing Jagged2. Favoring this hypothesis is the fact that membrane metalloproteases of the ADAM family are postulated to act at the cell surface is in favor of this hypothesis. KUZ-DN is shown not to be able to inhibit transactivation of a target gene of the Notch pathway induced by ligand binding to the receptor. In conclusion, mammalian Notch molecules are constitutively processed by a furin-like convertase as part of their normal maturation process, and this processing is required for cell surface expression of a heterodimeric functional receptor (Logeat, 1998).

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family, which encompasses a number of structurally homologous mitogens including EGF, TGF-alpha, vaccinia virus growth factor, amphiregulin, beta-cellulin and epiregulin. Like EGF, TGF-alpha and amphiregulin, HB-EGF binds to and stimulates the phosphorylation of the EGF receptor. HB-EGF is synthesized as a membrane-anchored precursor protein of 208 amino acids composed of signal peptide, heparin-binding, EGF-like, transmembrane and cytoplasmic domains. Although the membrane-anchored form of HB-EGF (proHB-EGF) is cleaved on the cell surface to yield a soluble growth factor of 75-86 amino acids, a considerable amount of proHB-EGF remains uncleaved on the cell surface. Importantly, proHB-EGF is not only a precursor of the soluble form but is also biologically active in itself; proHB-EGF forms a complex with both CD9 and integrin alpha3beta1, both localized at cell-cell attachment sites, and transduces biological signals in a nondiffusible manner to neighboring cells, as is known to occur for TGF-alpha and colony-stimulating factor. Moreover, although secreted mature HB-EGF is a potent mitogen for a number of cell types, the membrane-anchored form may act as a negative regulator of cell proliferation. Thus, the processing of the juxtamembrane domain of proHB-EGF to the soluble HB-EGF means the conversion of the mode of action of this growth factor from juxtacrine to paracrine. ProHB-EGF also acts as the specific receptor for diphtheria toxin (DT) and mediates the endocytosis of the receptor-bound toxin. Interestingly, proHB-EGF is cleaved rapidly to soluble HB-EGF by treatment with TPA, suggesting the involvement of a cellular signaling pathway involving protein kinase C (PKC). PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain (Izumi, 1998).

In order to investigate the role of ADAMs in early development a cDNA encoding a novel member of the ADAM family was cloned from a Xenopus laevis neurula stage library. X-ADAM 13 RNA is expressed during embryogenesis from the midblastula stage through tadpole stage 45. X-ADAM 13 is localized to somitic mesoderm and cranial neural crest cells during gastrulation, neurulation, and in tail bud stages. Sequence analyses of the X-ADAM 13 metalloprotease and disintegrin domains indicate that the protein is likely to be involved in both proteolytic and cell-adhesive functions. The X-ADAM 13 sequence is most closely related to that of mouse meltrin alpha, which is implicated in myoblast fusion. These data suggest that X-ADAM 13 may be involved in neural crest cell adhesion and migration as well as myoblast differentiation (Alfandari, 1997).

The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because of the physiological importance of this inflammatory cytokine. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases are involved. The enzyme that accomplishes shedding is TNF-alpha-converting enzyme, or TACE. It is a new member of a family of mammalian adamalysins (ADAMs) for which no physiological catalytic function has previously been identified. TACE is unique, with noteable sequence similarity to MADM, an enzyme implicated in myelin degradation, and to Kuzbanian (Black, 1997 and Moss, 1997).

Many membrane-bound proteins, including cytokines, receptors, and growth factors, are proteolytically cleaved to release a soluble form of their extracellular domain. The tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17) is a transmembrane metalloproteinase responsible for the proteolytic release or 'shedding' of several cell-surface proteins, including TNF and p75 TNFR. A TACE-reconstitution system has been established using TACE-deficient cells co-transfected with TACE and substrate cDNAs to study TACE function and regulation. Using the TACE-reconstitution system, two additional substrates of TACE, interleukin (IL)-1R-II and p55 TNFR, have been identified. Using truncations and chimeric constructs of TACE and another ADAM family member, ADAM-10, the function of the different domains of TACE were studied in three shedding activities. TACE must be expressed with its membrane-anchoring domain for phorbol ester-stimulated shedding of TNF, p75 TNFR, and IL-1R-II, but the cytoplasmic domain is not required for the shedding of these substrates. The catalytic domain of ADAM-10 could not be functionally substituted for that of TACE. IL-1R-II shedding requires the cysteine-rich domain of TACE as well as the catalytic domain, whereas TNF and p75 TNFR shedding requires only the tethered TACE catalytic domain (Reddy, 2000).

A recently identified gene encoding a metalloprotease-like, disintegrin-like, cysteine-rich protein (MDC) represents a candidate tumor suppressor gene for human breast cancer, based on its location within a minimal region of chromosome 17q21 previously defined by tumor deletion mapping. The MDC gene consists of 28 exons interrupted by relatively short introns. There are two forms of transcripts generated by alternative splicing. The more abundant form encodes a protein of 769 amino acids; the other, a previously described cDNA, encodes 524 amino acids. Exons 1a, 1b, 1c, 1d, and 2-7 encode a proprotein domain; exons 7-13 encode a metalloprotease-like domain; exons 14-17 encode a disintegrin domain; exons 18-22 encode a cysteine-rich domain, including an epidermal growth factor (EGF)-like repeat domain within exons 21 and 22; exon 23 encodes a transmembrane domain; and exons 24 and 25 encode a short cytoplasmic domain (Katagiri, 1995).

Skeletal muscle development involves the formation of multi-nucleated myotubes. This is thought to proceed by the induction of differentiation (acquisition of fusion competence) of myoblast cells. Myoblasts then aggregate and subsequently their plasma membranes fuse. Various membrane proteins including N- and M-cadherins, N- and V-CAMs and integrins participate in myotube formation, but the molecular mechanisms of muscle cell fusion are poorly understood. Three new, myoblast-expressed gene products have been identified (meltrin-alpha, beta and gamma), with homology to both viper hemorrhagic factors and fertilin (PH-30). Meltrin-alpha, a member of the metalloproteinase/disintegrin protein family, appears to be required for myotube formation. Involvement of a fertilin-related protein in myogenesis suggests that there are common mechanisms in gamete and myoblast fusion (Yagami-Hiromasa, 1995).

Metalloprotease-disintegrins are a family of membrane-anchored glycoproteins that have been implicated in diverse cellular processes, including fertilization and myoblast fusion, release of TNFalpha from the plasma membrane, and neurogenesis. The cloning of cDNAs encoding three full-length (xMDC9, xMDC11b, and xMDC13), and one partial (xMDC11a) metalloprotease-disintegrin from the amphibian Xenopus laevis is reported, and the analysis of their expression during early X. laevis development and in adult tissues. The most notable finding is the highly localized and specific expression pattern of xmdc11a at the tailbud stage in the cranial neural crest and in a subset of neural tube cells in the trunk region. In contrast, expression of the closely related xmdc11b is not detectable during the early stages of X. laevis development, and remains low in the adult tissues examined here. Distinct expression patterns are also observed for two other highly related X. laevis genes, xmdc13 and adam13. While adam13 is expressed in the somitic mesoderm and in neural crest cells, but not in adult testis, xmdc13 expression is low and ubiquitous in the developing embryo, but is clearly present in adult testis. Finally, xmdc9, the putative ortholog of human and mouse mdc9, was found at all stages of development, and in all tissues examined, suggesting a function that may be utilized by most or all cells (Cai, 1998).

Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. The release of the ectodomain and the enzymes involved have been investigated. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP-3, but not by inhibitors of other protease classes or by TIMP-2. This profile implicated MMPs or ADAMs as candidate sheddases. MMP-2, MMP-9 and MT1-MMP were excluded, but TACE, ADAM-10 and ADAM-9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE-deficient cells shedding was significantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.

Cadherins are critically involved in tissue development and tissue homeostasis. Neuronal cadherin (N-cadherin) is cleaved specifically by the disintegrin and metalloproteinase ADAM10 in its ectodomain. ADAM10 is not only responsible for the constitutive, but also for the regulated, shedding of this adhesion molecule in fibroblasts and neuronal cells directly regulating the overall levels of N-cadherin expression at the cell surface. The ADAM10-induced N-cadherin cleavage results in changes in the adhesive behaviour of cells and also in a dramatic redistribution of ß-catenin from the cell surface to the cytoplasmic pool, thereby influencing the expression of ß-catenin target genes. These data therefore demonstrate a crucial role of ADAM10 in the regulation of cell-cell adhesion and on ß-catenin signalling, leading to the conclusion that this protease constitutes a central switch in the signalling pathway from N-cadherin at the cell surface to ß-catenin/LEF-1-regulated gene expression in the nucleus (Reiss, 2005 ).

Epithelial-mesenchymal crosstalk is essential for tissue morphogenesis, but incompletely understood. Postnatal mammary gland development requires epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AREG), which generally must be cleaved from its transmembrane form in order to function. Since the transmembrane metalloproteinase ADAM17 can process AREG in culture and Adam17-/- mice tend to phenocopy Egfr-/- mice, the role of each of these molecules in mammary development was examined. Tissue recombination and transplantation studies reveal that EGFR phosphorylation and ductal development occur only when ADAM17 and AREG are expressed on mammary epithelial cells, whereas EGFR is required stromally, and that local AREG administration can rescue Adam17-/- transplants. Several EGFR agonists also stimulate Adam17-/- mammary organoid growth in culture, but only AREG is expressed abundantly in the developing ductal system in vivo. Thus, ADAM17 plays a crucial role in mammary morphogenesis by releasing AREG from mammary epithelial cells, thereby eliciting paracrine activation of stromal EGFR and reciprocal responses that regulate mammary epithelial development (Sternlicht, 2005).

Although Notch plays a crucial role in T cell development, regulation of Notch signaling in the thymus is not well understood. Kuzbanian, an ADAM protease, has been implicated in the cleavage of both Notch receptors and the Notch ligand, Delta. In this study it was shown that the expression of a dominant-negative form of Kuzbanian (dnKuz) leads to reduced TCRbeta expression in double-negative thymocytes and to a partial block between the double-negative to double-positive stages of development. These defects were rescued by overexpression of Delta-1 on thymocytes. Mixed chimeras showed a cell-autonomous block by dnKuz, but non-cell-autonomous rescue by Delta-1. This suggests that dnKuz impairs Notch signaling in receiving cells, and increasing Delta-1 on sending cells overcomes this defect. Interestingly, the expression of an activated form of Notch-1 rescued some, but not all, the defects in dnKuz Tg mice. These data suggest that multiple Notch-dependent steps in early thymocyte development require Kuzbanian, but differ in the involvement of other Notch signaling components (Manilay, 2005: full text of article).

Combinatorial mouse alleles for the secreted metalloproteases with thrombospondin motifs Adamts5, Adamts20 (bt), and Adamts9 result in fully penetrant soft-tissue syndactyly. Interdigital webs in double mutant Adamts5-/-;bt/bt mice had reduced apoptosis and decreased cleavage of the proteoglycan versican; however, the BMP-FGF axis, which regulates interdigital apoptosis was unaffected. BMP4 induced apoptosis, but without concomitant versican proteolysis. Haploinsufficiency of either Vcan or Fbln1, a cofactor for versican processing by ADAMTS5, led to highly penetrant syndactyly in bt mice, suggesting that cleaved versican was essential for web regression. The local application of an aminoterminal versican fragment corresponding to ADAMTS-processed versican, induced cell death in Adamts5-/-;bt/bt webs. Thus, ADAMTS proteases cooperatively maintain versican proteolysis above a required threshold to create a permissive environment for apoptosis. The data highlight the developmental significance of proteolytic action on the ECM, not only as a clearance mechanism, but also as a means to generate bioactive versican fragments (McCulloch, 2009).

Metalloproteinases and axon guidance

Contact-mediated axon repulsion by ephrins raises an unresolved question: these cell surface ligands form a high-affinity multivalent complex with their receptors present on axons, yet rather than being bound, axons can be rapidly repelled. Ephrin-A2 forms a stable complex with the metalloprotease Kuzbanian, involving interactions outside the cleavage region and the protease domain. Eph receptor binding triggers ephrin-A2 cleavage in a localized reaction specific to the cognate ligand. A cleavage-inhibiting mutation in ephrin-A2 delays axon withdrawal. These studies reveal mechanisms for protease recognition and control of cell surface proteins, and, for ephrin-A2, they may provide a means for efficient axon detachment and termination of signaling (Hattori, 2000).

Axons receive guidance information from extrinsic cues in their environment in order to reach their targets. In the frog Xenopus laevis, retinal ganglion cell (RGC) axons make three key guidance decisions en route through the brain. First, they cross to the contralateral side of the brain at the optic chiasm. Second, they turn caudally in the mid-diencephalon. Finally, they must recognize the optic tectum as their target. The matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) families are zinc (Zn)-dependent proteolytic enzymes. The latter functions in axon guidance, but a similar role has not yet been identified for the MMP family. Previous work has implicated metalloproteinases in the guidance decisions made by Xenopus RGC axons. To test specifically the importance of MMPs, two different in vivo exposed brain preparations were used in which RGC axons were exposed to an MMP-specific pharmacological inhibitor (SB-3CT), either as they reached the optic chiasm or as they extended through the diencephalon en route to the optic tectum. Interestingly, SB-3CT affects only two of the guidance decisions, with misrouting defects at the optic chiasm and tectum. Only at higher concentrations was RGC axon extension also impaired. These data implicate MMPs in the guidance of vertebrate axons, and suggest that different metalloproteinases function to regulate axon behaviour at distinct choice points: an MMP is important in guidance at the optic chiasm and the target, while either a different MMP or an ADAM is required for axons to make the turn in the mid-diencephalon (Hehr, 2005).

ADAMs and PTP-LAR degradation

Proteolytic processing and ectodomain shedding have been described for a broad spectrum of transmembrane proteins under both normal and pathophysiological conditions and has been suggested as one mechanism to regulate a protein's function. It has also been documented for the receptor-like protein tyrosine phosphatase PTP-LAR, induced by treating cells with the tumor promoter TPA or the calcium ionophor A23187. The epidermal growth factor receptor (EGFR) has been identified as both an association partner of PTP-LAR, that mediates phosphorylation of the latter, as well as an inducer of LAR-cleavage. Both overexpression of this kinase and stimulation of endogenous EGFR in various tumor cell lines have been shown to induce proteolytic processing of the catalytic LAR-P-subunit. In contrast to TPA-induced shedding of PTP-LAR, EGFR-mediated cleavage does not require PKC-activity. For both stimuli, however, processing of the P-subunit turns out to be dependent on the activation of the MAP kinases ERK1 and ERK2, and is completely abrogated upon pre-treating cells with Batimastat, indicating the involvement of a metalloproteinase in this pathway. Being strongly impaired in fibroblasts derived from ADAM-17/TACE-knockout-mice or tumor cells that express a dominant negative mutant of ADAM-17/TACE, cleavage of PTP-LAR is suggested to be mediated by this metalloproteinase. Paralleled by rapid reduction of cell surface-localized LAR-E-subunit, EGFR-induced cleavage could be shown to lead to degradation of the catalytic LAR-P-subunit, thereby resulting in a significantly reduced overall cellular phosphatase activity of PTP-LAR. These results for the first time identify a protein tyrosine phosphatase as a potential substrate of TACE and describe proteolytic processing of PTP-LAR as a means of regulating phosphatase activity downstream and thus under the control of EGFR-mediated signaling pathways (Ruhe, 2006).

ADAMs and the fertilization process

PH-30 is a sperm surface protein involved in sperm-egg fusion. It is composed of two subunits: alpha and beta, which are synthesized as precursors and subsequently processed during sperm development to yield the mature forms. The mature PH-30 alpha/beta complex resembles certain viral fusion proteins in membrane topology and predicted binding and fusion functions. Furthermore, the mature subunits are similar in sequence to one another and to a family of disintegrin domain-containing snake venom proteins. The sequences of the PH-30 alpha and beta precursor regions are also similar to one another, and to the precursors of snake venom metalloproteases and disintegrins. Reading from amino to carboxyl terminus, the alpha precursor region contains pro, metalloprotease, and disintegrin domains. The beta precursor region contains pro and metalloprotease domains. Residues diagnostic of a catalytically active metalloprotease are present in the alpha (but not the beta) precursor region. It has been proposed that the active sites of the PH-30 alpha and snake venom metalloproteases are structurally similar to that of astacin. PH-30, acting through its metalloprotease and/or disintegrin domains, could be involved in sperm development as well as sperm-egg binding and fusion. (Wolfsberg, 1993).

The ADAM 2 disintegrin loop, by virtue of its ability to bind integrins, is implicated in the process of fertilization in mammalian species. Since eggs from a variety of mammalian species express integrins on their surfaces, and since the integrin alpha6beta1 has been implicated in mouse sperm-egg binding, it is reasonable to propose that binding between the disintegrin domain of sperm ADAM 2 and the egg integrin accounts for the initial interaction between sperm and egg plasma membranes. ADAM 1 and ADAM 2 exists as a heterodimeric complex in some species; in one case, ADAM 1 does not possess a full disintegrin domain. ADAM 1 and 2 are likely to be intimately associated on the surface of all mammalian sperm. Three lines of indirect evidence support the hypothesis that ADAM 1 may be involved in membrane fusion: (1) all ADAM 1s cloned to date have a candidate fusion peptide, a hydrophobic segment in their cysteine-rich domains; (2) a synthetic form of the guinea pig ADAM 1 fusion peptide binds to membranes and induces fusion; and (3) another ADAM, ADAM 12, has been implicated in myoblast fusion. A working model has been proposed, wherein sperm-egg fusion occurs in two steps. The first step - binding - is fostered by the disintegrin domain of ADAM 2 (on the sperm) engaging an integrin on the egg plasm membrane. Next, a conformational change in the ADAM 1-2 complex exposes the fusion peptide of ADAM 1 so that it can bind, hydrophobically, to the egg layer. These events, in the context of a cluster of ADAM 1/2 complexes, would lead to the formation and opening of a fusion pore (Wolfsberg, 1996).

Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. Five distinct MDC proteins with integrin domains have been identified from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. The mRNA for xMDC16 is only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products are expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibit fertilization in a concentration-dependent manner, whereas xMDC16 peptides that are scrambled or have certain amino acid replacements in the predicted integrin-binding domain do not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibit fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 do not (Shilling, 1997).

Integrins can exist in different functional states with low or high binding capacity for particular ligands. The integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacts with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). The hypothesis was tested that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-transfected cells it was found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increase adhesion to laminin inhibit sperm binding. Conversely, treatments that inhibit laminin adhesion increase sperm binding. The ability of fluorescent beads coated with either fertilin beta or with the laminin E8 fragment to bind to eggs was examined. In Ca2+-containing media, fertilin beta beads bind to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 integrin subunit. In Ca2+-containing media, laminin E8 beads do not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibits fertilin bead binding, but does not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increases laminin E8 bead binding, and inhibits fertilin bead binding. These results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2) (Chen, 1999).

kuzbanian: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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