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Evolutionarily conserved developmental pathways
Secretion occurs in all cells, with relatively low levels in most cells and extremely high levels in specialized secretory cells, such as those of the pancreas, salivary, and mammary glands. How secretory capacity is selectively up-regulated in specialized secretory cells is unknown. This study found that the CrebA/Creb3-like family of bZip transcription factors functions to up-regulate expression of both the general protein machinery required in all cells for secretion and of cell type-specific secreted proteins. Drosophila CrebA directly binds the enhancers of secretory pathway genes and is both necessary and sufficient to activate expression of every secretory pathway component gene examined thus far. Microarray profiling reveals that CrebA also up-regulates expression of genes encoding cell type-specific secreted components. Finally, it was found that the human CrebA orthologues, Creb3L1 and Creb3L2, have the ability to up-regulate the secretory pathway in nonsecretory cell types (Fox, 2010).
This study provides evidence that the CrebA/Creb3-like bZIP transcription factors are direct and major regulators of secretory capacity. Drosophila CrebA directly activates high-level expression of secretory pathway component genes (SPCGs) through a site that is conserved among the enhancers of 34 CrebA-dependent SPCGs. Moreover, ectopic expression of CrebA in multiple tissues is sufficient to activate high-level expression of every SPCG tested. Microarray analysis indicates that CrebA is required for full expression of ~400 genes, including almost 200 implicated in secretion. The secretory target genes include general machinery required for secretion in all cells as well as cell type-specific secreted cargo, such as the cuticle proteins and mucins. Phenotypic characterization of CrebA mutant SGs revealed a range of expected secretory defects, including reduced luminal secretory content and a decrease in the size and frequency of apical secretory vesicles, as well as unexpected changes in organelle distribution. Active forms of the closest vertebrate orthologues Creb3L1 and Creb3L2 were found to activate the Drosophila SPCGs when expressed in embryos. Active Creb3L1 can also induce expression of multiple components of the secretory pathway when expressed in HeLa cells, a nonsecretory cell type (Fox, 2010).
CrebA is the single Drosophila member of the Creb3-like family of transcription factors that includes five different proteins in mammals (Creb3/Luman, Creb3L1/Oasis, Creb3L2/BBF2H7, Creb3L3/CrebH, and Creb3L4/Creb4) and two in worms (C27D6.4 and F57B10.1). This singularity means that the fly protein is likely to play a more pivotal role in the regulation of secretion because there is no possibility of compensation for its activity by other family members. Each member of the Creb3-like family has a unique expression pattern, with some overlap among family members. Creb3/Luman is most highly expressed in the brain, with expression detected in the liver, intestine, colon, and skeletal muscles. Creb3L1 is expressed in osteoblasts, prostate, pancreas, ovary, testis, the gut, lungs, kidney, and SGs. Creb3L2 is expressed in chondrocytes, heart, lung, liver, kidney, adrenal gland, bladder, submandibular gland, brain, ovary, pancreas, spleen, testis, and prostate. Creb3L3/CrebH is almost exclusively detected in the liver, whereas Creb3L4/Creb4 expression is elevated in the prostate, thymus, brain, pancreas, skeletal muscle, and peripheral leukocytes. Unlike the Drosophila and worm orthologues, all five members of the Creb3-like family are ER-bound transcription factors previously implicated as sensors in the unfolded protein response (UPR). Recently published phenotypes of the knockout mutations in each of the two genes most closely related to CrebA, Creb3L1, and Creb3L2 suggest a more physiological role for these genes during normal development, with a major defect being failure to secrete the extracellular matrix in the cell types expressing the highest levels of each gene (Murakami, 2009; Saito, 2009). These data support a model wherein one or more of the remaining members of the family may largely compensate for the loss of secretory capacity associated with the loss of any one family member. Indeed, findings that the expression of only a single Creb3-like family member in HeLa cells, a nonsecretory cell type, is sufficient to activate expression of multiple components of the secretory machinery further supports this hypothesis. Among the many secretory genes induced in HeLa cells by Creb3L1 are genes encoding multiple components of CopII vesicles: Sec16A, Sec23A, Sec24A, Sec24D, Sec31A, and Sar1A. The reduced expression of one or more of these genes could explain the ER trapping of ECM proteins observed with the loss of either Creb3L1 or Creb3L2 (Fox, 2010).
Microarray analysis of CrebA mutants revealed that CrebA up-regulates transcription of secretory cargo, specifically expression of multiple components of the insect cuticle, several mucin-like proteins (secreted highly-glycosylated proteins rich in serine and threonine), and multiple secreted proteins of unknown function. Although unexpected, this parallels the finding that mouse Creb3L1 directly up-regulates the type I collagen gene col1a1, a major secreted component of bone ECM (Murakami, 2009). The data also suggests that CrebA may function in parallel with tissue-specific regulators to control high-level expression of organ-specific cargo. An example is CG14756, which encodes an SG-specific secreted protein of unknown function. Loss of CrebA results in a 3.2-fold decrease in the expression of this gene based on the microarray analysis, but unlike the CrebA targets that show more general expression in all secretory tissues, expression of CG14756 could not be induced by CrebA in other cell types, which suggests the additional requirement for tissue-specific transcription factors for its activation. Indeed, expression of CG14756 is absolutely dependent on Fkh , and the region immediately upstream of CG14756 contains a good consensus Fkh binding site ~150 bp upstream of three clustered CrebA consensus binding sites. Thus, it is proposed that the CrebA/Creb3-like family enhances secretory capacity by coordinately up-regulating expression of the general secretory machinery and of tissue-specific secreted cargo, with the expression of cargo genes likely mediated through cooperation with tissue-specific factors (Fox, 2010).
More than 30% (116 of 383) of genes identified in the CrebA microarray experiments had GO terms associated with roles in the secretory pathway, and WoLF PSORT predictions suggested that more than half of the unknown targets are likely to have roles in secretion. Indeed, genes not implicated in the secretory pathway may, nonetheless, participate in secretion. Several of the ion channel/transporter genes have human orthologues known to function in secretory pathway organelles; for example, CG10449 (Drosophila catsup, human SLC39A7) encodes a Golgi-localized zinc transporter. Also, 26 of the target genes that did not have GO annotations have highly conserved human orthologues, several of which are involved in secretion. For example, CG4293 and CG7011 encode proteins similar to ERGIC2 and ERGIC3, respectively, which are proteins localized to the ER-Golgi intermediate compartment that function in protein folding and trafficking. Thus, it is likely that many of the newly identified CrebA target genes encode proteins that function in secretory organelles, highlighting the potential of the microarray studies to reveal new genes with key roles in the efficient production and delivery of products through the secretory pathway (Fox, 2010).
Altogether, these studies reveal that CrebA and its human orthologues Creb3L1 and Creb3L2 activate transcription of components that function at all steps in secretion. Coordinate up-regulation of secretory components by one (or a very few) transcription factors allows for easily adjustable levels of secretory capacity in a variety of cell types, as nicely exemplified in the Drosophila embryo, where levels of CrebA and corresponding SPCG expression correlate with the levels of secretory activity in the different tissues. Furthermore, microarray analysis combined with the recent studies of Creb3L1 and Creb3L2 in specialized cell types (osteoblasts and chondrocytes; Murakami, 2009; Saito, 2009) suggest that CrebA family proteins also up-regulate expression of tissue-specific secreted content, highlighting the significance of this protein family in secretory cell specialization and function (Fox, 2010).
Fox, R. M., Hanlon, C. D. and Andrew, D. J. (2010). The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity. J Cell Biol 191: 479-492. PubMed ID: 21041443
Murakami, T., et al. (2009). Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation. Nat Cell Biol 11: 1205-1211. PubMed ID: 19767743
Saito, A., Hino, S., Murakami, T., Kanemoto, S., Kondo, S., Saitoh, M., Nishimura, R., Yoneda, T., Furuichi, T., Ikegawa, S., Ikawa, M., Okabe, M. and Imaizumi, K. (2009). Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis. Nat Cell Biol 11: 1197-1204. PubMed ID: 19767744
Developmental Pathways conserved in Evolution
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