Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes

Patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) gene expression. We previously showed that SERCA2 downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). However, NRVM express three different PMA-sensitive PKC isoenzymes (PKCalpha, PKCepsilon, and PKCdelta), which may be differentially regulated and have specific functions in the cardiomyocyte. Therefore, in this study we used adenoviral vectors encoding wild-type (wt) and kinase-defective, dominant negative (dn) mutant forms of PKCalpha, PKCepsilon, and PKCdelta to analyze their individual effects in regulating SERCA2 gene expression in NRVM. Overexpression of wtPKCepsilon and wtPKCdelta, but not wtPKCalpha, was sufficient to downregulate SERCA2 mRNA levels, as assessed by Northern blotting and quantitative, real-time RT-PCR (69 +/- 7 and 61 +/- 9% of control levels for wtPKCepsilon and wtPKCdelta, respectively; P < 0.05 for each adenovirus; n = 8 experiments). Conversely, overexpression of all three dnPKCs appeared to significantly increase SERCA2 mRNA levels (dnPKCdelta > dnPKCepsilon > dnPKCalpha). dnPKCdelta overexpression produced the largest increase (2.8 +/- 1.0-fold; n = 11 experiments). However, PMA treatment was still sufficient to downregulate SERCA2 mRNA levels despite overexpression of each dominant negative mutant. These data indicate that the novel PKC isoenzymes PKCepsilon and PKCdelta selectively regulate SERCA2 gene expression in cardiomyocytes but that neither PKC alone is necessary for this effect if the other novel PKC can be activated.     

The protein phosphatase calcineurin determines basal parathyroid hormone gene expression

Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.     

Control of protein expression through mRNA stability in calcium signalling

Specific sequences (cis-acting elements) in the 3'-untranslated region (UTR) of RNA, together with stabilizing and destabilizing proteins (trans-acting factors), determine the mRNA stability, and consequently, the level of expression of several proteins. Such interactions were discovered initially for short-lived mRNAs encoding cytokines and early genes like c-jun and c-myc. However, they may also determine the fate of more stable mRNAs in a tissue and disease-dependent manner. The interactions between the cis-acting elements and the trans-acting factors may also be modulated by Ca(2+) either directly or via a control of the phosphorylation status of the trans-acting factors. We focus initially on the basic concepts in mRNA stability with the trans-acting factors AUF1 (destabilizing) and HuR (stabilizing). Sarco/endoplasmic reticulum Ca(2+) pumps, SERCA2a (cardiac and slow twitch muscles) and SERCA2b (most cells including smooth muscle cells), are pivotal in Ca(2+) mobilization during signal transduction. SERCA2a and SERCA2b proteins are encoded by relatively stable mRNAs that contain cis-acting stability determinants in their 3'-regions. We present several pathways where 3'-UTR mediated mRNA decay is key to Ca(2+) signalling: SERCA2a and beta-adrenergic receptors in heart failure, renin-angiotensin system, and parathyroid hormones. Other examples discussed include cytokines vascular endothelial growth factor, endothelin and endothelial nitric oxide synthase. Roles of Ca(2+) and Ca(2+)-binding proteins in mRNA stability are also discussed. We anticipate that these novel modes of control of protein expression will form an emerging area of research that may explore the central role of Ca(2+) in cell function during development and in disease.     

Cell death inhibiting RNA (CDIR) derived from a 3'-untranslated region binds AUF1 and heat shock protein 27

Regulators of programmed cell death were previously identified using a technical knockout genetic screen. Among the elements that inhibited interferon-gamma-induced apoptosis of HeLa cells was a 441-nucleotide fragment derived from the 3'-untranslated region (UTR) of KIAA0425, a gene of unknown function. This fragment was termed cell death inhibiting RNA (CDIR). Deletion and mutation analyses of CDIR were employed to identify the features required for its anti-apoptotic activity. Single nucleotide alterations within either copy of the duplicated U-rich motif found in the CDIR sequence abolished the anti-apoptotic activity of CDIR and altered its in vitro association with a protein complex. Further analysis of the CDIR-binding complex indicated that it contained heat shock protein 27 (Hsp27) and the regulator of mRNA turnover AUF1 (heterogeneous nuclear ribonucleoprotein D). In addition, recombinant AUF1 bound directly to CDIR. Furthermore, expression of another AUF1-binding RNA element, derived from the 3'-UTR of c-myc, inhibited apoptosis. We also demonstrate that the level and the stability of p21(waf1/Cip1/sdi1) mRNA, a target of AUF1 with anti-apoptotic activity, were increased in CDIR-transfected cells. The level of mRNA and protein of Bcl-2, another anti-apoptotic gene, containing an AUF1 binding site in its 3'-UTR was also increased in CDIR-transfected cells. Our data suggest that AUF1 regulates apoptosis by altering mRNA turnover. We propose that CDIR inhibits apoptosis by acting as a competitive inhibitor of AUF1, preventing AUF1 from binding to its targets.     

Regulation of the alternative splicing of sarcoplasmic reticulum Ca²⁺-ATPase1 (SERCA1) by phorbol 12-myristate 13-acetate (PMA) via a PKC pathway

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease with no established treatment to date. Small, cell-permeable molecules hold the potential to treat DM1. In this study, we investigated the association between protein kinase C (PKC) signaling and splicing of sarcoplasmic reticulum Ca(2+)-ATPase1 (SERCA1). Our aim was to clarify the mechanisms underlying the regulation of alternative splicing, in order to explore new therapeutic strategies for DM1. By assessing the splicing pattern of the endogenous SERCA1 gene in HEK293 cells, we found that treatment with phorbol 12-myristate 13-acetate (PMA) regulated SERCA1 splicing. Interestingly, treatment with PMA for 48 h normalized SERCA1 splicing, while treatment for 1.5h promoted aberrant splicing. These two responses showed dose dependency and were completely abolished by the PKC inhibitor Ro 31-8220. Furthermore, repression of PKCβII and PKCθ by RNAi mimicked prolonged PMA treatment. These results indicate that PKC signaling is involved in the splicing of SERCA1 and provide new evidence for a link between alternative splicing and PKC signaling.     

Role of AUF1 and HuR in the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK₁-F⁺ cells

Onset of metabolic acidosis leads to a rapid and pronounced increase in expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat renal proximal convoluted tubules. This adaptive response is modeled by treating a clonal line of porcine LLC-PK(1)-F(+) cells with an acidic medium (pH 6.9, 9 mM HCO(3)(-)). Measurement of the half-lives of PEPCK mRNA in cells treated with normal (pH 7.4, 26 mM HCO(3)(-)) and acidic medium established that the observed increase is due in part to stabilization of the PEPCK mRNA. The pH-responsive stabilization was reproduced in a Tet-responsive chimeric reporter mRNA containing the 3'-UTR of PEPCK mRNA. This response was lost by mutation of a highly conserved AU sequence that binds AUF1 and is the primary element that mediates the rapid turnover of PEPCK mRNA. However, siRNA knockdown of AUF1 had little effect on the basal levels and the pH-responsive increases in PEPCK mRNA and protein. Electrophoretic mobility shift assays established that purified recombinant HuR, another AU element binding protein, also binds with high affinity and specificity to multiple sites within the final 92-nucleotides of the 3'-UTR of the PEPCK mRNA, including the highly conserved AU-rich element. siRNA knockdown of HuR caused pronounced decreases in basal expression and the pH-responsive increases in PEPCK mRNA and protein. Therefore, basal expression and the pH-responsive stabilization of PEPCK mRNA in LLC-PK(1)-F(+) cells, and possibly in the renal proximal tubule, may require the remodeling of HuR and AUF1 binding to the elements that mediate the rapid turnover of PEPCK mRNA.     

Developmental regulation of RNA transcript destabilization by A + U-rich elements is AUF1-dependent

The developmental immaturity of neonatal phagocytic function is associated with decreased accumulation and half-life (t((1)/(2))) of granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in mononuclear cells (MNC) from the neonatal umbilical cord compared with adult peripheral blood. The in vivo t((1)/(2)) of GM-CSF mRNA is 3-fold shorter in neonatal (30 min) than in adult (100 min) MNC. Turnover of mRNA containing a 3'-untranslated region (3'-UTR) A + U-rich element (ARE), which regulates GM-CSF mRNA stability, is accelerated in vitro by protein fractions enriched for AUF1, an ARE-specific binding factor. The data reported here demonstrate that the ARE significantly accelerates in vitro decay of the GM-CSF 3'-UTR in the presence of either neonatal or adult MNC protein. Decay intermediates of the GM-CSF 3'-UTR are generated that are truncated at either end of the ARE. Furthermore, the t((1)/(2)) of the ARE-containing 3'-UTR is 4-fold shorter in the presence of neonatal (19 min) than adult (79 min) MNC protein, reconstituting developmental regulation in a cell-free system. Finally, accelerated ARE-dependent decay of the GM-CSF 3'-UTR in vitro by neonatal MNC protein is significantly attenuated by immunodepletion of AUF1, providing new evidence that this accelerated turnover is ARE- and AUF1-dependent.     

A putative proton binding site of plasma membrane H(+)-ATPase identified through homology modelling

We have used the 2.6 A structure of the rabbit sarcoplasmic reticulum Ca(2+)-ATPase isoform 1a, SERCA1a [Toyoshima, C., Nakasako, M., Nomura, H. and Ogawa, H. (2000) Nature 405, 647-655], to build models by homology modelling of two plasma membrane (PM) H(+)-ATPases, Arabidopsis thaliana AHA2 and Saccharomyces cerevisiae PMA1. We propose that in both yeast and plant PM H(+)-ATPases a strictly conserved aspartate in transmembrane segment (M)6 (D684(AHA2)/D730(PMA1)), and three backbone carbonyls in M4 (I282(AHA2)/I331(PMA1), G283(AHA2)/I332(PMA1) and I285(AHA2)/V334(PMA1)) comprise a binding site for H3O(+), suggesting a previously unknown mechanism for transport of protons. Comparison with the structure of the SERCA1a made it feasible to suggest a possible receptor region for the C-terminal auto-inhibitory domain extending from the phosphorylation and anchor domains into the transmembrane region.     

Phorbol ester differentially regulates oxytocin receptor binding activity in hypothalamic cultured neurons and astrocytes

Hypothalamic cultured neurons and astrocytes were used to investigate the cellular mechanisms underlying the oxytocin receptor-mediated downregulation through a possible involvement of protein kinase C (PKC). For this purpose, the effects of PKC activators, inhibitor and of OT on OT receptor binding activity were compared in both cultures. In neurons, phorbol-myristate-acetate (PMA), a potent PKC activator, increased the binding of an OT receptor antagonist whereas in astrocytes, a decrease was observed. Pre-treatment of the cells with bisindolylmaleimide (10(-4) M), a PKC inhibitor, prevented the PMA-induced up- and downregulation. In contrast, receptor downregulation resulting from treatment of both cells with OT (10(-9) M) was not affected by the PKC inhibitor. On the other hand, when PMA (10(-7) M) was tested along with OT (10(-9) M), a subsequent decrease in ligand binding was observed in astrocytes. In neurons, PMA attenuated the OT-induced downregulation. Structural analysis of neuron and astrocyte OT receptor mRNA by RT-PCR, subcloning and sequencing, demonstrated identical sequence to rat uterine receptor. In conclusion, these data suggest that activation of PKC has opposite effect on OT receptor binding activity in neurons and astrocytes but they do not support the involvement of PKC in the OT-induced downregulation.