cAMP Response Element Modulator Tau Research Articles

Cytochrome P450 metabolism of the post-lanosterol intermediates explains enigmas of cholesterol synthesis.

Cholesterol synthesis is among the oldest metabolic pathways, consisting of the Bloch and Kandutch-Russell branches. Following lanosterol, sterols of both branches are proposed to be dedicated to cholesterol. We challenge this dogma by mathematical modeling and with experimental evidence. It was not possible to explain the sterol profile of testis in cAMP responsive element modulator tau (Crem τ) knockout mice with mathematical models based on textbook pathways of cholesterol synthesis. Our model differs in the inclusion of virtual sterol metabolizing enzymes branching from the pathway. We tested the hypothesis that enzymes from the cytochrome P450 (CYP) superfamily can participate in the catalysis of non-classical reactions. We show that CYP enzymes can metabolize multiple sterols in vitro, establishing novel branching points of cholesterol synthesis. In conclusion, sterols of cholesterol synthesis can be oxidized further to metabolites not dedicated to production of cholesterol. Additionally, CYP7A1, CYP11A1, CYP27A1, and CYP46A1 are parts of a broader cholesterol synthesis network.

NR6A1 couples with cAMP response element binding protein and regulates vascular smooth muscle cell migration

Vascular smooth muscle cell (VSMC) migration is implicated in atherosclerosis and restenosis. Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is involved in regulating embryonic stem cell differentiation, reproduction, neuronal differentiation. Functional cooperation between cAMP response element modulator tau (CREMtau) and NR6A1 can direct gene expression in cells. cAMP response element binding protein (CREB) plays a key role in VSMC migration. In this study, we sought to determine whether CREB involved in NR6A1-modulated VSMC migration. VSMCs treated with platelet-derived growth factor-BB (PDGF-BB) displayed reduced mRNA and protein levels of NR6A1. Adenovirus-mediated expression of NR6A1 (Ad-NR6A1) could inhibit PDGF-BB- and serum-induced VSMC migration. The mRNA and protein expressions of secreted phosphoprotein 1 (SPP1) were down-regulated by NR6A1 overexpression. SPP1 promoter reporter activity was repressed by NR6A1. NR6A1 was found to physically couple with nuclear actin and the large subunit of RNA polymerase II. Furthermore, we showed that CREB interacted with NR6A1 in VSMCs. NR6A1 overexpression repressed cAMP response element (CRE) activity. ChIP assay revealed that NR6A1 bind to SPP1 promoter. Luciferase reporter assay showed that NR6A1 regulated SPP1 promoter activity via a putative CRE site. Adenovirus mediated local NR6A1 gene transfer attenuated stenosis after balloon-induced arterial injury in Sprague–Dawley rats. Taken together, this study provided experimental evidence that NR6A1 modulated SPP1 expression via its binding with CREB protein in VSMCs. We also revealed a NR6A1-CREB-SPP1 axis that serves as a regulatory mechanism for atherosclerosis and restenosis.

Sperm associated antigen 8 (SPAG8), a novel regulator of activator of CREM in testis during spermatogenesis

cAMP response element modulator (CREM)-mediated gene expression is an essential regulatory mechanism for germ cell differentiation. CREM and its coactivator in testis, ACT, activate the transcription of many essential genes for spermatogenesis. Sperm associated antigen 8 (SPAG8) is a testis-specific component that is expressed during germ cell differentiation. In this study, we found the pattern of SPAG8 expression largely overlapped with that of ACT during spermatogenesis and verified the association of SPAG8 with ACT. Furthermore, we showed that SPAG8 enhanced the transcriptional activation of ACT-mediated CREMτ by strengthening the binding of ACT to CREMτ. These results indicate that SPAG8 acts as a regulator of ACT and plays an important role in CREM-ACT-mediated gene transcription during spermatogenesis. Structured summaryMINT-7892874: CREM tau (uniprotkb:P27699-1) physically interacts (MI:0915) with ACT (uniprotkb:Q9WTX7) by anti tag coimmunoprecipitation (MI:0007)MINT-7892809: ACT (uniprotkb:Q9WTX7) physically interacts (MI:0915) with SPAG8 (uniprotkb:B9EKF1) by pull down (MI:0096)MINT-7892820, MINT-7892840, MINT-7892987, MINT-7892854: ACT (uniprotkb:Q9WTX7) physically interacts (MI:0915) with SPAG8 (uniprotkb:B9EKF1) by anti tag coimmunoprecipitation (MI:0007)

Functional cooperation between CREM and GCNF directs gene expression in haploid male germ cells

Cellular differentiation and development of germ cells critically depend on a coordinated activation and repression of specific genes. The underlying regulation mechanisms, however, still lack a lot of understanding. Here, we describe that both the testis-specific transcriptional activator CREMτ (cAMP response element modulator tau) and the repressor GCNF (germ cell nuclear factor) have an overlapping binding site which alone is sufficient to direct cell type-specific expression in vivo in a heterologous promoter context. Expression of the transgene driven by the CREM/GCNF site is detectable in spermatids, but not in any somatic tissue or at any other stages during germ cell differentiation. CREMτ acts as an activator of gene transcription whereas GCNF suppresses this activity. Both factors compete for binding to the same DNA response element. Effective binding of CREM and GCNF highly depends on composition and epigenetic modification of the binding site. We also discovered that CREM and GCNF bind to each other via their DNA binding domains, indicating a complex interaction between the two factors. There are several testis-specific target genes that are regulated by CREM and GCNF in a reciprocal manner, showing a similar activation pattern as during spermatogenesis. Our data indicate that a single common binding site for CREM and GCNF is sufficient to specifically direct gene transcription in a tissue-, cell type- and differentiation-specific manner.

Germ cell-specific cyclic adenosine 3',5'-monophosphate response element modulator expression in rodent and primate testis is maintained despite gonadotropin deficiency.

cAMP response element modulator (CREM) is an important component of the cAMP-mediated signaling pathway and is essential for differentiation of haploid male germ cells. In the rodent, testicular expression of CREM is believed to be controlled by FSH. We studied the expression pattern of CREM and gonadotropic control in the nonhuman primate and rodent testis. Adult cynomolgus monkeys (Macaca fascicularis) received daily either vehicle or the potent GnRH antagonist (ANT) cetrorelix for periods of 25 and 56 days. Rats were also exposed to vehicle or ANT for periods of 14 and 42 days. ANT treatment suppressed pituitary gonadotropin secretion, reduced testis size, and altered spermatogenesis. A rabbit polyclonal antibody raised against recombinant CREM tau and reacting with CREM alpha, -beta, -gamma, -tau1, and -tau2 at similar affinities was used for immunocytochemistry and Western blotting. CREM expression was seen in round spermatids, with highest levels during spermatogenic stages V-VII, but declined with progression of spermatid development in the primate. Similar observations were made for the rat testis. Thus, CREM expression was maximal at the onset of acrosome formation and was low or undetectable upon initiation of spermatid elongation in both species. A weak, but specific, CREM signal was seen in mid- to late pachytene spermatocytes and during meiotic division in both species. After ANT exposure, the germ cell- and stage-specific pattern of CREM expression was quantitatively retained at all time points and in both species. Northern and Western blot analysis confirmed the maintenance of testicular CREM expression despite 25 days of ANT treatment. A retrospective immunocytochemical analysis of rat testes 14 days posthypophysectomy revealed CREM signals in round spermatids. These findings demonstrate that the testicular expression of CREM is not entirely dependent on gonadotropic hormones but, rather, on the maturational stage of haploid round germ cells.

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.

Cell Cycle Regulation of Cyclin A Gene Expression by the Cyclic AMP-Responsive Transcription Factors CREB and CREM

Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated. We have investigated the role of the cyclic AMP (cAMP)-dependent signalling pathway in this cell cycle-dependent control. In human diploid fibroblasts (Hs 27), induction of cyclin A gene expression at G1/S is stimulated by 8-bromo-cAMP and suppressed by the protein kinase A inhibitor H89, which was found to delay S phase entry. Transfection experiments showed that the cyclin A promoter is inducible by activation of the adenylyl cyclase signalling pathway. Stimulation is mediated predominantly via a cAMP response element (CRE) located at positions -80 to -73 with respect to the transcription initiation site and is able to bind CRE-binding proteins and CRE modulators. Moreover, activation by phosphorylation of the activators CRE-binding proteins and CRE modulator tau and levels of the inducible cAMP early repressor are cell cycle regulated, which is consistent with the pattern of cyclin A inducibility by cAMP during the cell cycle. These results suggest that the CRE is, at least partly, implicated in stimulation of cyclin A transcription at G1/S.

Induction of CREM activator proteins in spermatids: down-stream targets and implications for haploid germ cell differentiation.

The cAMP-responsive element modulator (CREM) gene encodes a family of transcriptional regulators that bind to promoter sequences activated by increased intracellular cAMP levels. Both activators and repressors are generated by alternative splicing and alternative translational initiation. During the development of male germ cells, there is a switch in the transcripts generated by CREM. Specifically, from the prophase of meiosis, there is an increase in the CREM tau activator transcript. Here we present results showing that expression of the CREM activator protein is restricted to postmeiotic germ cells. We show that CREM tau is efficiently phosphorylated at a serine residue at position 117 by the protein kinase-A endogenous to germ cells, indicating that it constitutes a natural target of the adenylyl cyclase pathway during spermatogenesis. Phosphorylation of serine-117 turns CREM tau into a powerful activator. The rise in CREM tau protein coincides with the transcriptional activation of several genes. We show that CREM tau efficiently binds to CREs present in the promoters of these genes, suggesting that they could constitute down-stream targets of CREM. We have analyzed in more detail the regulation of one of these genes, the male germ cell-specific RT7. The RT7 promoter is cAMP inducible and activated by CREM tau in transfection assays. The RT7 promoter is efficiently transcribed in vitro with nuclear extracts from seminiferous tubules. CREM-specific antibodies block RT7 in vitro transcription, implicating a role for CREM tau in a cascade of transcriptional events during spermatogenesis.

Alternative usage of initiation codons in mRNA encoding the cAMP-responsive-element modulator generates regulators with opposite functions.

The cAMP-responsive-element modulator (CREM) gene encodes both antagonists (CREM alpha/beta/gamma) and an activator (CREM tau) of cAMP-responsive transcription by alternative splicing. In adult mouse brain a predominant 21-kDa protein, not corresponding to any previously characterized transcript, is detected with specific CREM antibodies. A developmental switch occurs in brain as expression changes at birth from CREM alpha/beta to the 21-kDa protein. We show that the 21-kDa protein corresponds to S-CREM (short CREM), a protein produced by the use of an internal AUG initiation codon in the CREM tau transcript. S-CREM shares with the other CREM proteins the basic DNA-binding and leucine-zipper dimerization domain. S-CREM functions as a transcriptional repressor of cAMP-induced transcription. Thus, two proteins with opposite functions are generated by alternative translation using two AUG codons within the same reading frame.