A coordinated phosphorylation cascade initiated by MSK1 directs RAR alpha recruitment to target gene promoters

The nuclear retinoic acid (RA) receptor alpha (RARα) is a transcriptional transregulator that controls the expression of specific gene subsets through binding at response elements and dynamic interactions with coregulators, which are coordinated by the ligand. Here, we highlighted a novel paradigm in which the transcription of RARα-target genes is controlled by phosphorylation cascades initiated by the rapid RA activation of the p38MAPK/MSK1 pathway. We demonstrate that MSK1 phosphorylates RARα at S369 located in the Ligand Binding Domain, allowing the binding of TFIIH and thereby phosphorylation of the N-terminal domain at S77 by cdk7/cyclin H. MSK1 also phosphorylates Histone H3 at S10. Finally, the phosphorylation cascade initiated by MSK1 is required for the recruitment of RARα/TFIIH complexes to response elements and subsequently for RARα target genes activation. Cancer cells characterized by a deregulated p38MAPK/MSK1 pathway, do not respond to RA, outlining the essential contribution of the RA-triggered phosphorylation cascade in RA signaling

Dynamic and combinatorial control of gene expression by nuclear retinoic acid receptors (RARs)

Nuclear retinoic acid receptors (RARs) are transcriptional regulators controlling the expression of specific subsets of genes in a ligand-dependent manner. The basic mechanism for switching on transcription of cognate target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes, the assembly of which is directed by the C-terminal ligand-binding domain of RARs. In addition to this scenario, new roles for the N-terminal domain and the ubiquitin-proteasome system recently emerged. Moreover, the functions of RARs are not limited to the regulation of cognate target genes, as they can transrepress other gene pathways. Finally, RARs are also involved in nongenomic biological activities such as the activation of translation and of kinase cascades. Here we will review these mechanisms, focusing on how kinase signaling and the proteasome pathway cooperate to influence the dynamics of RAR transcriptional activity

Efeitos in vitro do ácido retinóico em células de glioblastoma.

Malignant gliomas are highly invasive, rapidly proliferating tumors and present a poor prognosis. In this study, we investigated the potential effects of the retinoic acid (RA) on a high proliferative glioblastoma cell line, GL-15. The exposure to a single dose of t-RA reduces the cell growth, induces a transitory stage of the cell differentiation, and it finally leads to the apoptotic cell death depending on the RA concentration range and the time of exposure. We found that the GL-15 cells express constitutively the RARs isotypes a, b and g, and that RARa1/2, RARb2, and RARg2 expressions are induced by t-RA. These results suggest that the ratio of RAR isoforms may be a crucial element for inducing either efficient differentiaton or apoptotic effects in those cells. Furthermore, they suggest that the use of ligands specific to each receptor isotype might be relevant for further glioma therapies.Os gliomas malignos são tumores muito infiltrantes, cujas células proliferam rapidamente, e apresentam um prognóstico muito reservado. Neste estudo, investigamos o efeito em potencial do ácido retinóico (AR) sobre a linhagem de células de glioblastoma multiforme humano GL-15. A exposição a uma única dose de AR (1-10 mM) inibiu a proliferação celular, induziu uma diferenciação transitória e, finalmente, conduziu estas células à apoptose. Observamos que as células GL-15 expressam os isotipos dos RARs a, b e g, e que as isoformas RARa1/2, RARb2 e RARg2 são induzidas pelo AR. Estes resultados sugerem que a relação entre a expressão das diferentes isoformas de RARs pode ser um elemento fundamental para a indução seja de uma diferenciação completa, seja de apoptose das células de glioblastoma, e que o uso de ligantes específicos a cada isotipo de receptor pode vir a ser um elemento importante para terapias futuras de gliomas

Androgen receptor phosphorylation at serine 308 and serine 791 predicts enhanced survival in castrate resistant prostate cancer patients

We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR<sub>94</sub>), 308 (pAR<sub>308</sub>), 650(pAR<sub>650</sub>) and 791(pAR<sub>791</sub>). No correlations with clinical parameters were observed for pAR<sub>94</sub> or pAR<sub>650</sub> in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR<sub>308</sub> is significantly associated with a longer time to disease specific death (p= 0.011) and high pAR<sub>791</sub> expression significantly associated with a longer time to disease recurrence (p= 0.018) in HNPC tumours and longer time to death from disease recurrence (p= 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p= 0.022) and low proliferating tumours (p= 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer

Increase of poly(ADP-ribose) polymerase mRNA levels during TPA-induced differentiation of human lymphocytes

AbstractThe non-mitogenic stimulation of human peripheral blood mononuclear cells (PBMC) with low concentrations of the phorbol ester 12-O-tetradecanoylphorbol 13-acatate (TPA) caused a progressive increase in the percent fraction of the cells that were positive for the early activating antigen CD69. At the same time, it caused a progressive increase in the steady-state levels of poly(ADP-ribose) polymerase (pADPRP) transcripts. A further increase in TPA concentration, while inducing the maximal expression of the levels of CD69 activating surface antigen, both in the presence or in the absence of proliferative activity, did not evoke any additional hightening of pADPRP mRNA levels. Time course of PBMC stimulation with a non-mitogenic dose or TPA showed an early increase in the accumulation of pADPRP mRNA, which changed at 8-16 h. and remained high for several days thereafter. On the basis of these data, we suggest flat the increase in pADPRP mRNA may be associated with the commitment of human lymphocytes from a quiescent (G0) to an activated (G1) state

Molecular Mechanisms of Retinoid Receptors in Diabetes-Induced Cardiac Remodeling

Diabetic cardiomyopathy (DCM), a significant contributor to morbidity and mortality in diabetic patients, is characterized by ventricular dysfunction, in the absence of coronary atherosclerosis and hypertension. There is no specific therapeutic strategy to effectively treat patients with DCM, due to a lack of a mechanistic understanding of the disease process. Retinoic acid, the active metabolite of vitamin A, is involved in a wide range of biological processes, through binding and activation of nuclear receptors: retinoic acid receptors (RAR) and retinoid X receptors (RXR). RAR/RXR-mediated signaling has been implicated in the regulation of glucose and lipid metabolism. Recently, it has been reported that activation of RAR/RXR has an important role in preventing the development of diabetic cardiomyopathy, through improving cardiac insulin resistance, inhibition of intracellular oxidative stress, NF-κB-mediated inflammatory responses and the renin-angiotensin system. Moreover, downregulated RAR/RXR signaling has been demonstrated in diabetic myocardium, suggesting that impaired RAR/RXR signaling may be a trigger to accelerate diabetes-induced development of DCM. Understanding the molecular mechanisms of retinoid receptors in the regulation of cardiac metabolism and remodeling under diabetic conditions is important in providing the impetus for generating novel therapeutic approaches for the prevention and treatment of diabetes-induced cardiac complications and heart failure

Dynamic Interaction of TTDA with TFIIH Is Stabilized by Nucleotide Excision Repair in Living Cells

Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER

Both XPD alleles contribute to the phenotype of compound heterozygote xeroderma pigmentosum patients

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in the rare recessive genetic disorder xeroderma pigmentosum (XP). Many XP patients are compound heterozygotes with a “causative” XPD point mutation R683W and different second mutant alleles, considered “null alleles.” However, there is marked clinical heterogeneity (including presence or absence of skin cancers or neurological degeneration) in these XPD/R683W patients, thus suggesting a contribution of the second allele. Here, we report XP patients carrying XPD/R683W and a second XPD allele either XPD/Q452X, /I455del, or /199insPP. We performed a systematic study of the effect of these XPD mutations on several enzymatic functions of TFIIH and found that each mutation exhibited unique biochemical properties. Although all the mutations inhibited the nucleotide excision repair (NER) by disturbing the XPD helicase function, each of them disrupted specific molecular steps during transcription: XPD/Q452X hindered the transactivation process, XPD/I455del disturbed RNA polymerase II phosphorylation, and XPD/199insPP inhibited kinase activity of the cdk7 subunit of TFIIH. The broad range and severity of clinical features in XP patients arise from a broad set of deficiencies in NER and transcription that result from the combination of mutations found on both XPD alleles