cAMP Response Element Modulator Isoforms Research Articles

The function of cAMP responsive element modulator (CREM) in PDGF induced proliferation of vascular smooth muscle cells

The transcription factor CREM plays an important role in cAMP mediated gene expression. Here we want to clarify the role of CREM in vasculature particularly with regard to the regulation of VSMCs proliferation, using CREM (CKO) and CREM isoform ICER (IKO) deficient mice.CKO mice showed an increase of neointima formation associated with a higher proliferation rate of aortic VSMCs under platelet‐derived growth factor (PDGF) treatment. The PDGF alpha receptor (Pdgfra) promoter was inducible by cAMP and mRNA was upregulated in aortae and PDGF‐treated primary VSMCs of CKO mice. In untreated IKO‐VSMCs the proportion of proliferating cells was higher as compared to WT‐VSMCs, while no differences were observed under PDGF treatment. Induction of cAMP by forskolin reduced the proliferation rate of both IKO and WT‐VSMCs under PDGF treatment.Accordingly, ICER was linked to the elevated proliferation of VSMCs under non‐stimulated conditions, however, other CREM isoforms than ICER inhibit PDGF‐mediated proliferation of VSMCs possibly by modulation of cAMP‐dependent Pdgfra gene regulation. (supported by DFG)

Transcriptional Activation of the cAMP-responsive Modulator Promoter in Human T Cells Is Regulated by Protein Phosphatase 2A-mediated Dephosphorylation of SP-1 and Reflects Disease Activity in Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with numerous abnormalities recorded at the cellular, molecular, and genetic level. Expression of the basic leucine zipper transcription factor cAMP-responsive element modulator (CREM)α was reported to be abnormally increased in T cells from SLE patients. CREMα suppresses IL-2 and T cell receptor ζ chain gene transcription by direct binding to the respective promoters. Here, we show that increased CREM expression is the result of enhanced promoter activity. DNA binding analyses reveal direct binding of transcription factor specificity protein-1 (SP-1) to the CREM promoter resulting in enhanced transcriptional activity and increased CREM expression. Protein phosphatase 2A is known to activate SP-1 through dephosphorylation at its serine residue 59. Our results show that nuclei from SLE T cells contain lower levels of Ser(59)-phosphorylated SP-1 protein and a stronger SP-1 binding to the CREM promoter. We conclude that protein phosphatase 2A accounts for enhanced SP-1 dephosphorylation at Ser(59) in SLE T cells. More importantly, CREM promoter activity mirrors reliably disease activity in SLE patients, underscoring its potential role as a biomarker for the prediction of flares in SLE patients.

Functional Characterization of Male Germ Cell‐Specific CREM Isoforms

Due to alternative promoter usage, splicing, and translational initiation, expression of the cAMP-responsive element modulator (CREM) gene results in the production of functionally different CREM proteins with either activating or repressing potential on target gene expression. Recently, we demonstrated 2 novel isoforms (CREM-2-F-G-H-Ib and CREM-2-G-H-Ib) in various germ cell types during normal and impaired human spermatogenesis. In contrast to known isoforms, these exhibit a transactivation domain but lack a kinase-inducible domain (KID) domain resulting in a disruption of the open reading frame. In the present study, we functionally analyzed these isoforms. Investigation of both in vitro and in vivo expressed proteins from human testis RNA suggests that a novel upstream open reading frame in exon 2 is translated from isoform CREM-2-F-G-H-Ib, giving rise to a full-length protein. Furthermore, in both isoforms, usage of downstream adeninethymine-guanines (ATGs) for translation initiation could be observed. Sequence-specific DNA binding of CREM isoforms was confirmed by electrophoretic mobility shift assays. Luciferase reporter gene assays in cells transfected with novel CREM cDNAs demonstrated that protein kinase A dependent stimulation was inhibited by coexpression of CREM-2-F-G-H-Ib but not of CREM-2-G-H-Ib.

Crem Activator Isoforms in Normal and Impaired Human Spermatogenesis Analyzed by Real Time RT-PCR

cAMP responsive element modulator (CREM) activator isoforms are involved in mammalian spermatogenesis and spermiogenesis. CREM proteins are highly expressed in postmeiotic germ cells of rodents and primates. Homozygous CREM inactivated mice exhibit round spermatid maturation arrest. The lack of CREM expression at both the mRNA and protein levels is associated with spermatid maturation arrest in infertile patients. Using real-time RT-PCR, we have examined the levels of CREM activator isoform mRNAs: CREMtheta1, CREMtheta2 and CREMt2 + Ex-gamma in gametogenic and interstitial cell fractions from normal human testis, in homogenized tissue samples from spermatogenic arrest and from testicular tumors. We have shown for the first time the presence of CREM activator isoform containing exon gamma (CREMtau2 + Exgamma) in normal human spermatogenesis. Among the three CREM isoforms, CREMtheta1 was expressed in its highest level in the male gonads. In comparison, CREMtheta2 mRNA was significantly less suggesting that the P3 promoter is much more active in human testis than the P4 promoter. Minimal-nill levels of mRNA for either of the CREM activator isoforms were detected in lymphocytes or in gonadal tissues from patients with SCOS (Sertoli Cell Only Syndrome). This data underlines the significance of CREMtheta1 isoform in the regulation of transcription during post-meiotic germ cell differentiation.

Novel cyclic adenosine 3',5'-monophosphate (cAMP) response element modulator theta isoforms expressed by two newly identified cAMP-responsive promoters active in the testis.

cAMP signaling contributes to the control of the developmental progression of germ cells during the spermatogenic cycle. Genes regulated by cAMP include those encoding transcription factors such as the cAMP-responsive element modulator (CREM). The disruption of CREM gene expression in crem null mice results in arrest of spermatogenesis and infertility. The transcriptional control of the CREM gene is attributed to two promoters, P1 and P2. The P1 promoter constitutively activates the synthesis of messenger RNAs encoding activator (tau) and repressor (alpha) forms of CREM, whereas the cAMP-responsive P2 promoter activates the formation of messenger RNAs encoding the inducible cAMP early repressor. Here we report the identification of two additional promoters in the CREM gene, P3 and P4, that in the rat testis encode two novel transcriptional activator CREM isoforms, termed CREM theta1 and CREM theta2, respectively. Notably, the P3 and P4 promoters are activated by cAMP-dependent protein kinase, thereby providing cAMP-regulated transcription of CREM activators in addition to the established cAMP-regulated inducible cAMP early repressor. Analysis ex vivo of CREM gene expression in temporally staged segments of the seminiferous tubule during the spermatogenic cycle shows that the activities of the P1, P3, and P4 promoters are independently regulated. Our identification of the cAMP-activated P3 and P4 promoters that direct expression of the novel theta1 and theta2 activator isoforms of CREM brings further insight into the complex expression of the CREM gene during germ cell development and may have implications in understanding the control of fertility.

Requirement for cAMP-response element (CRE) binding protein/CRE modulator transcription factors in thyrotropin-induced proliferation of dog thyroid cells in primary culture.

In several cell types, mostly of epithelial origin, activation of the cAMP pathway triggers DNA synthesis and cell division. Regulation of gene expression by cAMP involves phosphorylation by pyruvate kinase A and activation of cAMP-response element binding protein (CREB)/CRE modulator (CREM) transcription factors which bind DNA to CRE sites. On the other hand, several CREM isoforms are transcriptional repressors, such as the inducible cAMP early repressor (ICER) transcription factors, which are synthesized from an intronic promoter of the CREM gene. This study investigated the potential role of CREB/CREM transcription factors in the cAMP mitogenic pathway, using an experimental model of epithelial cells in primary culture, i.e. dog thyroid cells stimulated by thyroid-stimulating hormone (TSH). In response to TSH, CREB/CREM transcription factors were phosphorylated on the serine residue of the pyruvate kinase A consensus site. In addition, the synthesis of ICER mRNAs was strongly induced by TSH. This transient upregulation of ICER expression correlated with increased protein levels. It was restricted to the cAMP pathway, as neither epidermal growth factor nor phorbol myristate acetate, which are potent mitogens for dog thyroid cells, induced ICER expression. On the other hand, increased expression of ICER mRNAs was not detected in dog thyroids chronically stimulated by TSH in vivo. The requirement for CREB/CREM transcription factors in the mitogenic effect of TSH was assessed by transfecting expression vectors encoding CREM repressors into dog thyrocytes in order to interfere with CRE-mediated gene transcription. The ectopic expression of ICER Igamma or CREM alpha isoforms inhibited DNA replication in dog thyrocytes stimulated by TSH. This inhibitory effect was dependent on the ability of CREM repressors to form dimers but did not involve their DNA-binding capacity. Together these results show that CREB/CREM transcription factors are tightly regulated, at the transcriptional and post-translational levels, by TSH in dog thyroid cells, and provide clear evidence that their activity is required for the cAMP-dependent proliferation of cells in primary culture. Moreover, the transient induction of ICER transcription factors during mitogenic stimulation by TSH raises questions about the role of these potent repressors of CRE-dependent transcription as timers of cellular proliferation.

Dog CREM Transcription Factors: Cloning, Tissue Distribution, and Identification of New Isoforms

CREM (cAMP Response Element Modulator) transcription factors are involved in the cAMP-dependent transcriptional regulation of CRE-containing genes. Multiple CREM transactivators and repressors are generated from a single gene by alternative splicings and use of an alternative intronic promoter. Here we report the cloning and sequencing of the full-length dog CREM cDNA, corresponding to the CREMτα splice variant. Amino acid sequence identity with mouse and human orthologs reached 94.5% and 91.0% respectively. Using the RNAse Protection Assay (RPA) method with three distinct probes, we analyzed the expression of the various CREM transcripts in several dog tissues. We showed that CREM transcription factors have a restricted tissue distribution and that the ratio between activators and repressors varies considerably from one tissue to another. Moreover, we amplified, by RT-PCR, a cDNA that corresponds to two new CREM isoforms and confirmed, by RPA experiments, the presence of these mRNAs in dog thyroid and in other tissues. These transcripts result from splicing of the γ domain and encode potential CREM transactivators (CREMταγ and CREMτ2αγ).

Differential Activation of Viral and Cellular Promoters by Human T-cell Lymphotropic Virus-1 Tax and cAMP-responsive Element Modulator Isoforms

We have previously proposed that cAMP-responsive element-binding protein (CREB) activity is stimulated by human T-cell lymphotropic virus-1 (HTLV-1) Tax through two mechanisms that are differentially dependent upon CREB phosphorylation. We have tested this model by examining how Tax affects transcriptional activation mediated by the cAMP-responsive element (CRE) modulator (CREM). The CREM proteins are highly homologous to CREB, particularly in their DNA-binding domains and the kinase-inducible domain (KID), a region that interacts with the coactivator CREB-binding protein (CBP) in a phosphorylation-dependent manner. Despite this similarity, most CREM isoforms are transcriptional repressors. CREMalpha lacks the glutamine-rich domains found in CREB that are essential for transcriptional activation. We show that the normally repressive CREMalpha activates the HTLV-1 and cellular CREs in the presence of Tax; activation of the viral element is phosphorylation-independent, and activation of the cellular CRE is phosphorylation-dependent. CREMDelta(C-G) lacks both the KID and the glutamine-rich regions. This isoform activates the HTLV-1 long terminal repeat in a phosphorylation-independent manner, but does not activate the cellular CRE. This study suggests that Tax, interacting with the basic/zipper region of CREM, recruits CBP to the viral promoter. Tax activation of the cellular CRE depends on the KID and its ability to interact with CBP in a phosphorylation-dependent manner.

CAMP-response element modulator tau is a positive regulator of testis angiotensin converting enzyme transcription.

Testis angiotensin-converting enzyme (ACE) is a unique form of ACE, only produced by male germ cells, and results from a testis-specific promoter found within the ACE gene. We have investigated the role of cAMP-response element modulator (CREM)tau in testis ACE transcription. In gel shift experiments, testes nuclear proteins retard an oligonucleotide containing the cAMP-response element (CRE) found at position -55 in the testis ACE promoter. Anti-CREM antibody supershifts this complex. Competitive gel shift shows that recombinant CREM tau protein and testis nuclear proteins have a similar specificity of binding to the tests ACE CRE. Functional analysis using in vitro transcription and transfection studies also demonstrate that CREM tau protein is a transcriptional activator of the testis ACE promoter. Western blot analysis identifies CREM tau protein in the protein-DNA complex formed between nuclear proteins and the testis ACE CRE motif. This analysis also identified other CREM isoforms in the gel-shifted complex, which are thought to be CREM tau 1/2, CREM alpha/beta, and S-CREM. These data indicate that CREM tau isoforms play an important role as a positive regulator in the tissue-specific expression of testis ACE.

Role of transcription factors CREB and CREM in cAMP-regulated transcription during spermatogenesis

The cAMP response element binding protein (CREB) and the cAMP-responsive element modulator (CREM) are cyclically expressed at high levels during spermatogenesis. Cyclical expression of CREB and CREM in germ and somatic Sertoli cells correlates with the fluctuations in cAMP signaling induced by the pituitary gonadotropic hormones FSH and LH both during sexual maturation of the testis and during the 12-day cycles of spermatogenesis that occur in the adult testis. CREB and CREM are expressed at different times during the spermatogenic cycle, undergo programmed sequential switches from activator to repressor isoforms by mechanisms of alternative exon splicing and promoter usage, and are autoregulated by cAMP signaling in opposing directions. cAMP response elements located in the promoter of the CREB gene upregulate the expression of activator CREBs, whereas cAMP autoregulatory response elements in the internal promoter of the CREM gene induce expression of repressor CREM isoforms. The complex mechanisms for the regulation of the expression of CREB and CREM in the testis appear to reflect critical adaptations for regulating key target genes essential for the development of germ cells.