Protein Kinase C Regulates Internal Initiation of Translation of the GATA-4 mRNA following Vasopressin-induced Hypertrophy of Cardiac Myocytes

Abstract

GATA-4 is a key member of the GATA family of transcription factors involved in cardiac development and growth as well as in cardiac hypertrophy and heart failure. Our previous studies suggest that GATA-4 protein synthesis may be translationally regulated. We report here that the 518-nt long 5-untranslated region (5-UTR) of the GATA-4 mRNA, which is predicted to form stable secondary structures (؊65 kcal/mol) such as to be inhibitory to cap-dependent initiation, confers efficient translation to monocistronic reporter mRNAs in cell-free extracts. Moreover, uncapped GATA-4 5-UTR containing monocistronic reporter mRNAs continue to be well translated while capped reporters are insensitive to the inhibition of initiation by cap-analog, suggesting a capindependent mechanism of initiation. Utilizing a dicistronic luciferase mRNA reporter containing the GATA-4 5-UTR within the intercistronic region, we demonstrate that this leader sequence confers functional internal ribosome entry site (IRES) activity. The activity of the GATA-4 IRES is unaffected in trans-differentiating P19CL6 cells, however, is strongly stimulated immediately following arginine-vasopressin exposure of H9c2 ventricular myocytes. IRES activity is then maintained at submaximal levels during hypertrophic growth of these cells. Supraphysiological Ca 2؉ levels diminished stimulation of IRES activity immediately following exposure to vasopressin and inhibition of protein kinase C activity utilizing a pseudosubstrate peptide sequence blocked IRES activity during hypertrophy. Thus, our data suggest a mechanism for GATA-4 protein synthesis under conditions of reduced global cap-dependent translation, which is maintained at a submaximal level during hypertrophic growth and point to the regulation of GATA-4 IRES activity by sarco(ER)-reticular Ca 2؉ stores and PKC.

Vasopressin V1a and V1b Receptors: From Molecules to Physiological Systems

“…Our study showed that AVP-induced cardiomyocyte hypertrophy was suppressed in primary culture from V1aR-KO mice, confirming the involvement of the V1a receptor in cell proliferation (221). In the cellular model of cardiomyocyte hypertrophy, AVP promotes translation of the transcription factor GATA-4 through PKC in H9c2 ventricular myocytes (455). Since the V1a receptor is widely expressed in various malignant cells, including neuroendocrine tumors (377) and small cell lung cancer (SCLC) (319), the AVP/V1a receptor axis may represent a novel target for new tumor therapies.…”

mTORC2 Activity Is Elevated in Gliomas and Promotes Growth and Cell Motility via Overexpression of Rictor

“…The primer sequences are as follows: forward primer, 5 ¶-GAGAAGTATCCTCAAACATTTG-3 ¶; reverse primer, 5 ¶-GCATTATGCTGAGTGATATCCCGCTCAGGATTCAGCAGATG-TATC-3 ¶. An antisense riboprobe was used to visualize 18S rRNA levels (27). For quantitative reverse transcription PCR, total RNA was reverse transcribed with random primers using the RETROscript Kit from Ambion.…”

Combined Tyrosine and Serine/Threonine Kinase Inhibition by Sorafenib Prevents Progression of Experimental Pulmonary Hypertension and Myocardial Remodeling

“…Arginine vasopressin is an endogenous peptidic hormone and a stimulator of hypertrophy in cardiomyocytes 19,20 as well as in H9c2 cells. 21,22 Hypertrophy was induced by treatment of confluent H9c2 cells in 24 wells (2ϫ10 4 cells) with arginine vasopressin in Dulbecco's modified Eagle's medium with 0.25% fetal calf serum. Medium was replaced daily.…”

“…For the subsequent kinase inhibition experiments, the concentration of arginine vasopressin was set to an excess of 1 mol/L as described previously. 21, 22 An ATPGlow cell viability assay (Promega) demonstrated cell viability rates of Ͼ85% at sorafenib or imatinib concentrations Ͻ1 mol/L. At concentrations Ͼ1 mol/L, however, viability rates decreased to 4% and 0% (sorafenib) and were 114% and 56% (imatinib) at 3 and 10 mol/L, respectively.…”