USF2 inhibits C/EBP-mediated transcriptional regulation of the RIIβ subunit of cAMP-dependent protein kinase

BACKGROUND: Cyclic AMP-dependent protein kinase (PKA) plays a central role in regulation of energy metabolism. Upon stimulation of testicular Sertoli cells by follicle stimulating hormone (FSH), glycolysis is activated to increase the production of nutrients for the germ cells, and a new regulatory subunit of cAMP-dependent protein kinase, RIIβ, is induced. We have previously shown that production of the transcription factor C/EBPβ is rapidly increased by FSH and cAMP in primary Sertoli cell cultures, and that C/EBPβ induces the RIIβ promoter. RESULTS: In this work we show that USF1, USF2 and truncated USF isoforms bind to a conserved E-box in the RIIβ gene. Interestingly, overexpression of USF2, but not USF1, led to inhibition of both cAMP- and C/EBPβ-mediated induction of RIIβ. Furthermore, Western blots show that a novel USF1 isoform is induced by cAMP in Sertoli cells. CONCLUSIONS: These results indicate that the expression of various USF isoforms may be regulated by cAMP, and that the interplay between USF and C/EBPβ is important for cAMP-mediated regulation of RIIβ expression. The counteracting effects of USF2 and C/EBPβ observed on the RIIβ promoter is in accordance with the hypothesis that C/EBP and USF play opposite roles in regulation of glucose metabolism

Interplay between the heterotrimeric G-protein subunits Gαq and Gαi2 sets the threshold for chemotaxis and TCR activation

<p>Abstract</p> <p>Background</p> <p>TCR and CXCR4-mediated signaling appears to be reciprocally regulated pathways. TCR activation dampens the chemotactic response towards the CXCR4 ligand CXCL12, while T cells exposed to CXCL12 are less prone to subsequent TCR-activation. The heterotrimeric G proteins G_αqand G_αi2have been implicated in CXCR4-signaling and we have recently also reported the possible involvement of G_αqin TCR-dependent activation of Lck (Ngai et al., Eur. J. Immunol., 2008, 38: 32083218). Here we examined the role of G_αqin migration and TCR activation.</p> <p>Results</p> <p>Pre-treatment of T cells with CXCL12 led to significantly reduced Lck Y394 phosphorylation upon TCR triggering indicating heterologous desensitization. We show that knockdown of G_αqsignificantly enhanced basal migration in T cells and reduced CXCL12-induced SHP-1 phosphorylation whereas G_αi2knockdown inhibited CXCL12-induced migration.</p> <p>Conclusion</p> <p>Our data suggest that G_αi2confers migration signals in the presence of CXCL12 whereas G_αqexerts a tonic inhibition on both basal and stimulated migrational responses. This is compatible with the notion that the level of G_αqactivation contributes to determining the commitment of the T cell either to migration or activation through the TCR.</p

Protein kinase A antagonist inhibits β-catenin nuclear translocation, c-Myc and COX-2 expression and tumor promotion in ApcMin/+ mice

<p>Abstract</p> <p>Background</p> <p>The adenomatous polyposis coli (APC) protein is part of the destruction complex controlling proteosomal degradation of β-catenin and limiting its nuclear translocation, which is thought to play a gate-keeping role in colorectal cancer. The destruction complex is inhibited by Wnt-Frz and prostaglandin E₂(PGE₂) - PI-3 kinase pathways. Recent reports show that PGE₂-induced phosphorylation of β-catenin by protein kinase A (PKA) increases nuclear translocation indicating two mechanisms of action of PGE₂on β-catenin homeostasis.</p> <p>Findings</p> <p>Treatment of <it>Apc</it>^Min/+mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS) selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas. Immunohistochemical characterization of intestines from treated and control animals revealed that expression of β-catenin, β-catenin nuclear translocation and expression of the β-catenin target genes c-Myc and COX-2 were significantly down-regulated upon Rp-8-Br-cAMPS treatment. Parallel experiments in a human colon cancer cell line (HCT116) revealed that Rp-8-Br-cAMPS blocked PGE₂-induced β-catenin phosphorylation and c-Myc upregulation.</p> <p>Conclusion</p> <p>Based on our findings we suggest that PGE₂act through PKA to promote β-catenin nuclear translocation and tumor development in <it>Apc</it>^Min/+mice <it>in vivo</it>, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.</p

FOXC2 Is a Winged Helix Gene that Counteracts Obesity, Hypertriglyceridemia, and Diet-Induced Insulin Resistance

AbstractObesity, hyperlipidemia, and insulin resistance are common forerunners of type 2 diabetes mellitus. We have identified the human winged helix/forkhead transcription factor gene FOXC2 as a key regulator of adipocyte metabolism. Increased FOXC2 expression, in adipocytes, has a pleiotropic effect on gene expression, which leads to a lean and insulin sensitive phenotype. FOXC2 affects adipocyte metabolism by increasing the sensitivity of the β-adrenergic-cAMP-protein kinase A (PKA) signaling pathway through alteration of adipocyte PKA holoenzyme composition. Increased FOXC2 levels, induced by high fat diet, seem to counteract most of the symptoms associated with obesity, including hypertriglyceridemia and diet-induced insulin resistance—a likely consequence hereof would be protection against type 2 diabetes

Localization of a Novel Human A-Kinase-Anchoring Protein, hAKAP220, during Spermatogenesis

AbstractUsing a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS–PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s)

Mice with Disrupted Type I Protein Kinase A Anchoring in T Cells Resist Retrovirus-Induced Immunodeficiency

Type I protein kinase A (PKA) is targeted to the TCR-proximal signaling machinery by the A-kinase anchoring protein ezrin and negatively regulates T cell immune function through activation of the C-terminal Src kinase. RI anchoring disruptor (RIAD) is a high-affinity competitor peptide that specifically displaces type I PKA from A-kinase anchoring proteins. In this study, we disrupted type I PKA anchoring in peripheral T cells by expressing a soluble ezrin fragment with RIAD inserted in place of the endogenous A-kinase binding domain under the lck distal promoter in mice. Peripheral T cells from mice expressing the RIAD fusion protein (RIAD-transgenic mice) displayed augmented basal and TCR-activated signaling, enhanced T cell responsiveness assessed as IL-2 secretion, and reduced sensitivity to PGE2- and cAMP-mediated inhibition of T cell function. Hyperactivation of the cAMP–type I PKA pathway is involved in the T cell dysfunction of HIV infection, as well as murine AIDS, a disease model induced by infection of C57BL/6 mice with LP-BM5, a mixture of attenuated murine leukemia viruses. LP-BM5–infected RIADtransgenic mice resist progression of murine AIDS and have improved viral control. This underscores the cAMP–type I PKA pathway in T cells as a putative target for therapeutic intervention in immunodeficiency diseases.Peer reviewe

PCM4EU and PRIME-ROSE:Collaboration for implementation of precision cancer medicine in Europe

Background: In the two European Union (EU)-funded projects, PCM4EU (Personalized Cancer Medicine for all EU citizens) and PRIME-ROSE (Precision Cancer Medicine Repurposing System Using Pragmatic Clinical Trials), we aim to facilitate implementation of precision cancer medicine (PCM) in Europe by leveraging the experience from ongoing national initiatives that have already been particularly successful. Patients and methods: PCM4EU and PRIME-ROSE gather 17 and 24 partners, respectively, from 19 European countries. The projects are based on a network of Drug Rediscovery Protocol (DRUP)-like clinical trials that are currently ongoing or soon to start in 11 different countries, and with more trials expected to be established soon. The main aims of both the projects are to improve implementation pathways from molecular diagnostics to treatment, and reimbursement of diagnostics and tumour-tailored therapies to provide examples of best practices for PCM in Europe. Results: PCM4EU and PRIME-ROSE were launched in January and July 2023, respectively. Educational materials, including a podcast series, are already available from the PCM4EU website (http://www.pcm4eu. eu). The first reports, including an overview of requirements for the reimbursement systems in participating countries and a guide on patient involvement, are expected to be published in 2024. Conclusion: European collaboration can facilitate the implementation of PCM and thereby provide affordable and equitable access to precision diagnostics and matched therapies for more patients.</p