CREB function in various tissues

Resting thymocytes contain predominantly unphosphorylated (inactive) CREB, which is rapidly activated by phosphorylation on Ser 119 following thymocyte activation. T-cell development is normal in transgenic mice that express a dominant-negative form of CREB (CREBA119, with alanine at position 119) under the control of the T-cell-specific CD2 promoter/enhancer. In contrast, thymocytes and T cells from these animals display a profound proliferative defect characterized by markedly decreased interleukin-2 production, G1 cell-cycle arrest and subsequent apoptotic death in response to a number of different activation signals. This proliferative defect is associated with the markedly reduced induction of c-jun, c-fos, Fra-2 and FosB following activation of the CREBA119 transgenic thymocytes. It is proposed that T-cell activation leads to the phosphorylation and activation of CREB, which in turn is required for normal induction of the transcription factor AP1 and subsequent interleukin-2 production and cell-cycle progression (Barton, 1996).

In chickens, PKA and PKC are involved in intracellular signaling during feather morphogenesis. Protein kinase C (PKC) immunoreactivity increases in the whole layer of developing dermis. This is followed by a gradual and highly localized decrease of PKC expression immediately beneath each forming feather germ. In contrast, cAMP response element binding protein (CREB) is ubiquitously expressed in both epithelium and mesenchyme. From stage 29 on, phosphorylated CREB (P-CREB), reflecting the activity of protein kinase A (PKA), begins to be seen in placode but not in interplacode epithelia. P-CREB is also expressed in bud mesenchyme transiently between stages 33 and 36, but not in the interbud mesenchyme. PKA activators and PKC inhibitors can expand a feather bud domain by enhancing dermal condensation, while PKC activators and PKA inhibitors can expand interbud domains. Neural cell adhesion molecule (N-CAM) is involved in dermal condensation. Activation of PKA causes diffused expression of N-CAM in mesenchyme while activation of PKC causes the disappearance of N-CAM in precondensed mesenchymal regions. A model of how the well-concerted PKA and PKC signaling may be involved in the formation and size regulation of dermal condensation is presented (Noveen, 1995).

A number of studies over the last several years have demonstrated a crucial role for TGF-beta in epithelial and mesenchymal differentiation during development of the embryonic palate. Molecular mechanism(s) of signal transduction responsible for eliciting these responses remain unresolved. Since cAMP signaling also modulates the same tissue differentiation in the developing palate and palate-derived cells, it was hypothesized that TGF-beta activity may be mediated through cAMP-inducible pathways and CREB. The effects of TGF-beta were examined on activation of CREB. An examination was made of the ability of TGF-beta-treated murine embryonic palate mesenchymal (MEPM) cells to phosphorylate CREB on the amino acid residue serine 133, phosphorylation of which is indispensable for transcriptional activation. TGF-beta treatment leads to increased phosphorylation of CREB ser-133 in a time- and dose-dependent manner. Inhibition of serine-threonine phosphatases by okadaic acid enhances but does not prolong this response. TGF-beta fails to induce the activity of protein kinase A (PKA), a known CREB kinase. Inhibition of either PKA or calcium/calmodulin kinase II (CaMK II) does not abrogate phosphorylation of CREB by TGF-beta. TGF-beta treatment also does not induce phosphorylation of mitogen-activated protein kinases, neither erk-1 nor erk-2, on tyrosine 185, suggesting that these kinases do not mediate CREB phosphorylation by TGF-beta. TGF-beta has no effect on CREB binding to known CREB DNA consensus recognition sequences: CRE and TRE. Together, these data suggest an alternative or novel CREB kinase in MEPM cells through which TGF-beta acts to induce CREB ser-133 phosphorylation and subsequent activation of CRE-containing genes (Potchinsky, 1997).

LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Besides its posttranscriptional regulation, LAP/C/EBP beta mRNA is modulated during liver regeneration. Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrates that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. Two sites have been identified that are important for specific complex formation within this region. CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that shown for the CREB consensus sequence. These sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. The protein kinase A pathway not only stimulates the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. After two-thirds hepatectomy a functional link exists between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. Since several pathways control CREB phosphorylation, these results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions (Niehof, 1997).

The expression of the genes encoding the hormones glucagon, insulin, somatostatin, and pancreatic polypeptide in the endocrine islets of the pancreas is regulated in a cell-specific manner, defining four distinct cellular phenotypes, respectively termed A-, B-, D-, and F-cells. Binding of nuclear proteins to cognate DNA sequences within cis-acting regulatory elements mediates the transcriptional events that result in the cell-specific activation or repression of gene expression. SMS-UE is a pancreatic islet D-cell specific enhancer element that regulates the expression of the somatostatin gene and contains two interdependent domains: A and B. Domain A of the SMS-UE is a DNA enhancer sequence that is identical to that bound by the ubiquitously distributed CCAAT box-binding protein alpha-CBF, a transcription factor that regulates the expression of the human chorionic gonadotrophin alpha-subunit gene. In contrast the B-domain binds an islet cell-specific protein with characteristics similar to those of Isl-1, a transcriptional activator protein that binds to the E2 enhancer of the rat insulin-1 gene. The SMS-UE binds transcription factor CREB but not CREM, the close homolog of CREB, on a site either adjacent to or overlapping the 3' end of domain B. The carboxyl-terminal bZIP domain of CREB binds to the cAMP response element of the somatostatin gene but is not sufficient for binding to the SMS-UE. CREB/SMS-UE binding requires stabilization by a region of the protein located within the transactivation domain (Vallejo, 1992).

CREB function in gonads

The cAMP/protein kinase A signaling pathway activates the cAMP-responsive transcription factor CREB. A unique alternative RNA splicing event occurs during the development of germ cells in the testis, resulting in a translational switch from an mRNA encoding activator CREB to an mRNA encoding novel inhibitor CREB (I-CREB) isoforms. Alternative splicing of an additional exon into the CREB mRNA in mid to late pachytene spermatocytes results in the premature termination of translation and consequent downstream reinitiation of translation producing I-CREBs. The I-CREBs down-regulate cAMP-activated gene expression by inhibiting activator CREB from binding to cAMP response elements. Further, the developmental stage-specific expression of I-CREBs in germ cells of the seminiferous tubules correlates with the cyclical down-regulation of activator CREB, suggesting that I-CREBs repress expression of the cAMP-inducible CREB gene as well as other genes transiently induced by cAMP during the 12-day cycle of spermatogenesis (Walker, 1996).

The somatic Sertoli cells of the testis are major targets for FSH and are important for the regulation of spermatogenesis. The binding of FSH to Sertoli cells activates the cAMP-dependent protein kinase A signaling pathway, resulting in phosphorylation of the cAMP response element-binding protein (CREB), which is required to transactivate genes containing cAMP response elements (CREs). The addition of forskolin to cultured primary Sertoli cells results in the phosphorylation of CREB within 2-5 min. Phospho-CREB levels remain elevated with continued forskolin stimulation, but fall by 60% within 5 min after the removal of forskolin. In addition, 8-bromo-cAMP induces CREB RNA accumulation in the Sertoli cells. There is a conserved 300-base pair region of the CREB promoter surrounding the transcription start site that is required for both basal and cAMP-inducible expression of the CREB gene. This region of the promoter contains three Sp1-binding sites flanking the transcription initiation site and two CREs located 65 and 85 base pairs downstream of the transcription initiation site. The Sp1 motifs bind Sp1 in Sertoli extracts and contribute to basal promoter activity. The CREs bind CREB and are essential for cAMP induction of CREB gene transcription. These findings support the model of FSH- and cAMP-mediated CREB autoregulation of its own promoter and may explain the dramatic stage-specific oscillations in Sertoli cells of CREB messenger RNA levels during the 12-day cycles of spermatogenesis in rat seminiferous tubules (Walker, 1995).

There is an alternatively spliced testicular CREM isoform, CREM delta C-G, lacking four exons including those encoding the protein kinase A-regulated phosphorylation domain and the flanking glutamine-rich transcriptional activation domains. CREM delta C-G retains exons that encode the basic-leucine zipper (bZIP) DNA-binding domain, binds to cAMP response elements (CREs), and competitively inhibits binding of CREB and CREM to CREs. Expression of CREM delta C-G inhibits transcription of a CRE-containing chloramphenicol acetyltransferase reporter plasmid induced by endogenous CREB. Antiserum to CREM detects CREM delta C-G in elongated spermatids from rat testis. These observations indicate that CREM delta C-G is a unique form of a competitive negative regulator of CREB-mediated gene transcription expressed in a maturation-dependent manner in haploid germ cells. The developmental specificity of CREM delta C-G suggests that it may play a role in transcriptional regulation during spermatogenesis (Walker, 1994).

The transcription of the transferrin (Tf) gene is induced by follitropin via cAMP in rat Sertoli cells. The cAMP-responsive-element-binding protein (CREB) interacts on the proximal region II (PRII) of the human Tf promoter. The PRII region is identified as essential for cAMP inducibility of the Tf promoter and contains a CCAAT box. This unexpected result led to a study of the relation that exists between CREB and the PRII site. In the liver, CCAAT/enhancer-binding (C/EBP) proteins act at the PRII site. Although these factors are absent in Sertoli cells, their overexpression in Sertoli cells disturbs basal and induced transcription. The Ka of CREB bZIP (254-327), a deleted form of CREB, for a CRE site and for a PRII site are similar, although the derived kinetics are different: higher Ka and Kd of CREB for the PRII site are found, when compared with the CRE site. It is thought that the binding of CREB to the PRII site is stabilized by CREB-binding protein (CBP) or by chicken-ovalbumin-upstream-promoter transcription factor (COUP-TF) binding to PRI site near to PRII. However, the overexpression of CBP in Sertoli cells does not potentiate the basal and cAMP-stimulated activity of CREB. In basal and cAMP-stimulated conditions, COUP-TF appeared to repress transcription. These results demonstrate a direct action of CREB on hTf promoter, which is antagonized by COUP-TF and may explain the transcriptional regulation of Tf by follitropin, via cAMP (Suire, 1996).

CREB and immediate response genes

Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes. Short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways. At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)-p62TCF, which mediates part of the UV-induced transcriptional response. More that one pathway is required for full UV responsiveness of c-fos. Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), it has been found that UVC-induced transcriptional activation depends also on the CRE at position -60 of the c-fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not (at least not decisively) participate in this signaling pathway to CREB and ATF-1, since it is not p38/RK/HOG-1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route of UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2-p62TCF pathway (Iordanov, 1997).