Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation.

Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel-Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22(phox)-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22(phox)-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22(phox)-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22(phox) reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

Novel mechanisms of protein synthesis in diabetic nephropathy--role of mRNA translation.

Ambient protein levels are affected by both synthesis and degradation. Synthesis of a protein is regulated by transcription and messenger RNA (mRNA) translation. Translation has emerged as an important site of regulation of protein expression during development and disease. It is under the control of distinct factors that regulate initiation, elongation and termination phases. Regulation of translation occurs via signaling reactions, guanosine diphosphate-guanosine triphosphate binding and by participation of non-coding RNA species such as microRNA. Recent work has revealed an important role for translation in hypertrophy, matrix protein synthesis, elaboration of growth factors in in vivo and in vitro models of diabetic nephropathy. Studies of translation dysregulation in diabetic nephropathy have enabled identification of novel therapeutic targets. Translation of mRNA is a fertile field for exploration in investigation of kidney disease.

Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase.

Angiotensin II (Ang II) exerts contractile and trophic effects in glomerular mesangial cells (MCs). One potential downstream target of Ang II is the protein kinase Akt/protein kinase B (PKB). We investigated the effect of Ang II on Akt/PKB activity in MCs. Ang II causes rapid activation of Akt/PKB (5-10 min) but delayed activation of phosphoinositide 3-kinase (PI3-K) (30 min). Activation of Akt/PKB by Ang II was not abrogated by the PI3-K inhibitors or by the introduction of a dominant negative PI3-K, indicating that in MCs, PI3-K is not an upstream mediator of Akt/PKB activation by Ang II. Incubation of MCs with phospholipase A2 inhibitors also blocked Akt/PKB activation by Ang II. AA mimicked the effect of Ang II. Inhibitors of cyclooxygenase-, lipoxyogenase-, and cytochrome P450-dependent metabolism did not influence AA-induced Akt/PKB activation. However, the antioxidants N-acetylcysteine and diphenylene iodonium inhibited both AA- and Ang II-induced Akt/PKB activation. Dominant negative mutant of Akt/PKB or antioxidants, but not the dominant negative form of PI3-K, inhibited Ang II-induced protein synthesis and cell hypertrophy. These data provide the first evidence that Ang II induces protein synthesis and hypertrophy in MCs through AA/redox-dependent pathway and Akt/PKB activation independent of PI3-K.

Activation of renal signaling pathways in db/db mice with type 2 diabetes.

BACKGROUND: Altered regulation of signaling pathways may contribute to the pathogenesis of renal disease. We examined renal cortical signaling pathways in type 2 diabetes. METHODS: The status of renal cortical signaling pathways was examined in control and db/db mice with type 2 diabetes in the early phase of diabetic nephropathy associated with renal matrix expansion and albuminuria. RESULTS: Tyrosine phosphorylation of renal cortical proteins was increased in diabetic mice. Renal cortical activities of phosphatidylinositol 3-kinase (PI 3-kinase) in antiphosphotyrosine immunoprecipitates, Akt (PKB), and ERK1/2-type mitogen-activated protein (MAP) kinase activities were significantly augmented sixfold (P < 0.01), twofold (P < 0.0003), and sevenfold (P < 0.001), respectively, in diabetic mice compared with controls. A part of the increased renal cortical PI 3-kinase activity was due to insulin receptor activation, as PI 3-kinase activity associated with beta chain of the insulin receptor was increased nearly fourfold (P < 0.0235). Additionally, the kinase activity of the immunoprecipitated insulin receptor beta chain was augmented in the diabetic renal cortex, and tyrosine phosphorylation of the insulin receptor was increased. In the liver, activities of PI 3-kinase in the antiphosphotyrosine immunoprecipitates and Akt also were increased threefold (P < 0.05) and twofold (P < 0.0002), respectively. However, there was no change in the hepatic insulin receptor-associated PI 3-kinase activity. Additionally, the hepatic ERK1/2-type MAP kinase activity was inhibited by nearly 50% (P < 0.01). CONCLUSIONS: These studies demonstrate that a variety of receptor signaling pathways are activated in the renal cortex of mice with type 2 diabetes, and suggest a role for augmented insulin receptor activity in nephropathy of type 2 diabetes.

Insulin regulation of protein translation repressor 4E-BP1, an eIF4E-binding protein, in renal epithelial cells.

BACKGROUND: Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) that follows release of eIF4E from a heterodimeric complex by phosphorylation of its inhibitory binding protein, 4E-BP1. We examined insulin regulation of 4E-BP1 phosphorylation in murine proximal tubular epithelial cells. METHODS AND RESULTS: Insulin (1 nmol/L) increased de novo protein synthesis by 58 +/- 11% (P < 0.001). Insulin also augmented 4E-BP1 phosphorylation and phosphatidylinositol 3-kinase (PI 3-kinase) activity in antiphosphotyrosine immunoprecipitates. This could be prevented by PI 3-kinase inhibitors, Wortmannin, and LY294002. Insulin also activated Akt that lies downstream of PI 3-kinase. Rapamycin abrogated 4E-BP1 phosphorylation in response to insulin, suggesting involvement of mammalian target of rapamycin (mTOR), a kinase downstream of Akt. Insulin-stimulated phosphorylation of 4E-BP1 was also inhibited by PD098059, implying involvement of Erk-1/-2 mitogen-activated protein (MAP) kinase. An increase in Erk-1/-2 type MAP kinase activity by insulin was directly confirmed in an immunokinase assay and was found to be PI 3-kinase dependent. CONCLUSIONS: In proximal tubular epithelial cells, insulin augments 4E-BP1 phosphorylation, which is PI 3-kinase and mTOR dependent. The requirement for Erk-1/-2 MAP kinase activation for 4E-BP1 phosphorylation by insulin suggests a cross-talk between PI 3-kinase and Erk-1/-2-type MAP kinase pathways.

Potential role of insulin-like growth factor binding protein-4 in the uncoupling of bone turnover in multiple myeloma.

Decreased bone formation plays an important role in the development of lytic lesions during the late stage of multiple myeloma (MM). Release of insulin-like growth factor binding protein-4 (IGFBP4) by tumour cells adjacent to bone may inhibit IGF-I-stimulated osteoblast growth and contribute to decreased bone formation. The present study demonstrates that the human MM cell line, ARH-77, expresses IGFBP4 and, to a lesser extent, IGFBP6 mRNA and protein. IGFBP4 expression in myeloma cells may be modulated by cytokines released by stromal cells and T cells in the microenvironment. We tested the effect of recombinant interferon-gamma (INF) on IGFBP4 expression in ARH-77. INF increased IGFBP4 mRNA and protein levels at 12 h, with a decline to baseline by 24 h. In contrast, IGFBP4 was not regulated in response to IL-6, TNF-alpha, PDGF BB, bFGF, TGF-beta or the cAMP agonist, forskolin. In other systems. IGFBP4 may also be regulated post-transcriptionally by a protease that is activated by IGF-I or -II. Conditioned medium from ARH-77 cultures incubated with IGF-I or -II for up to 24 h failed to demonstrate proteolytic activity. Proteolysis was also not observed when conditioned medium containing exogenous rhIGFBP4 was incubated with IGF-I or -II under cell-free conditions. To determine if human myeloma tumours also express IGFBP4, total RNA was isolated from four tumour biopsies. All samples expressed detectable levels of IGFBP4 mRNA. These findings indicate that interferon-gamma may indirectly modulate bone formation via the the release of tumour-derived IGFBP4. suggesting that the immune system may influence bone turnover in MM. Failure of myeloma cells to release protease activity may promote IGFBP4 accumulation in the microenvironment during tumour growth.

Annexin V inhibits protein kinase C activity via a mechanism of phospholipid sequestration.

In this study, we assessed the role of annexin V, a Ca2+-dependent phospholipid-binding protein, as a regulator of protein kinase C (PKC) and characterized its mechanism of inhibition. Several mutants obtained by oligonucleotide site-directed mutagenesis were tested in vitro on PKC activity in cytosolic fractions from Jurkat cells and on purified PKCalpha. Annexin V inhibited phosphorylation of annexin II by endogenous PKC and phosphorylation of myelin basic protein by PKCalpha. In both systems, the use of single Ca2+-binding-site mutants of annexin V led to a partial reversal of inhibition, and the Ca2+-binding site located in the first domain of annexin V was found to have the most important role. An increase in the number of mutated Ca2+-binding sites led to a greater loss of inhibition. These results corroborated those showing the progressive loss of binding of these mutants to phospholipid liposomes. In conclusion, we show that PKC inhibition by annexin V is the consequence of a mechanism involving phospholipid sequestration by annexin V, and that the Ca2+-binding site located in domain 1 of annexin V plays a predominant role in this process. In addition, we show that the R122AIK site, which may act analogously to a PKC-inhibitory pseudosubstrate site, is not involved in PKC inhibition, and that a peptide corresponding to the C-terminal tail of annexin V inhibits PKC activity but to a lesser extent than annexin V itself.

Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells.

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-beta (TGF-beta) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-beta stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-beta is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF-I and TGF-beta differentially regulate IGFBP-3, IGFBP-4, and IGFBP-5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF-I.

Interaction between insulin-like growth factor-I and insulin-like growth factor-binding proteins in TC-1 stromal cells.

IGF-I and -II play an important role in regulating bone formation. Bone marrow stromal cells, particularly those with osteoblast-like features, may act in concert with osteoblasts to increase IGF-I and -II levels in the bone microenvironment. Local bioavailability of IGFs, however, is modulated by IGF binding proteins (IGFBPs). We have previously demonstrated that murine TC-1 stromal cells constitutively secrete IGF-I and IGFBPs. In the present study, we determined the phenotype of these cells and used them as a model to explore the effect of IGFBPs on IGF-I-induced mitogenesis. The effect of IGF-I on IGFBPs expressed by TC-1 was also determined. When grown under conditions that promote osteogenic differentiation, TC-1 cells showed high alkaline phosphatase activity and mRNA levels, weakly expressed osteocalcin mRNA, and formed mineralized bone-like nodules. TC-1 cells expressed IGF-I and IGF-II mRNAs, while other stromal phenotypes preferentially expressed IGF-I. IGF-I stimulated TC-1 DNA synthesis in a dose-dependent manner and this effect was inhibited by recombinant IGFBP-1 and -4. Since IGF-I may regulate IGFBP production, the effect of IGF-I on IGFBPs expressed by TC-1 cells was determined. IGF-I increased the abundance of IGFBP-3, -4 and -5 in TC-1 conditioned medium; this correlated with induction of IGFBP-3 mRNA, but not with that of IGFBP-4 or -5 mRNAs. The findings demonstrate that most stromal cells express IGF-I which may act in an autocrine and/or paracrine fashion. The local effects of IGF-I, however, may be blocked by IGFBP-1 or -4. IGF-I regulates the relative abundance of IGFBPs in stromal cells which, in turn, may influence IGF-I-mediated effects on bone remodeling.

Inhibitory effect of annexin V on protein kinase C activity in mesangial cell lysates.

Annexin V belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of the protein kinase C (PKC) activity. This study examines the effect of annexin V on the in vitro PKC activity in cultured mesangial cells using histone H1, the peptide [Ser25]PKC-(19-31), or endogenous proteins as substrates. The SDS/PAGE pattern of 32P-labeled mesangial proteins showed that the calcium-independent PKC [(n+a)PKC] phosphorylated several proteins from 70 kDa to 40 kDa and 22 kDa to 15 kDa. Three additional proteins from 34 kDa to 29 kDa, including annexin I and its proteolytic forms, were detected after activation of calcium-dependent PKC (cPKC). Increasing concentrations of annexin V did not alter the phosphorylation of (n+a)PKC substrates. By contrast, specific phosphorylation of proteins and annexin I by cPKC, was reduced in a dose-dependent manner. Addition of high concentration of calcium and phosphatidylserine did not reverse the inhibitory effect of annexin V. Annexin V also inhibited the phosphorylation of histone H1 or peptide [Ser25]PKC-(19-31) by cPKC. Moreover, removal of annexin V from cytosols increased the annexin I phosphorylation by these isoforms. From these results, we propose that annexin V may regulate the signal-transduction pathway involving the activation of cPKC, as they act in vitro as an inhibitor of these kinases.

Forskolin mimics TSH action on the expression of protein kinase C isozymes in pig thyroid cell cultures.

In porcine thyroid cell cultures, phospholipid-dependent protein kinases (PKCs) have the same characteristics as intact glands. The overall PKC activity, presence of PKC isozymes, chromatographic pattern and endogenous substrates specificity were not modified during the two-day culture period. Three PKC isozymes (cPKC epsilon, nPKC epsilon and aPKC zeta) were identified by immunoblot analysis in the two subcellular fractions: cytosol and particulate extract, both in intact glands and two-day-old cultures. In cells cultured for two days in the presence of TSH (0.1 mU/ml), the overall PKC activity was stimulated (ca. 200%) in the two compartments. This stimulation was parallel to the increase in protein expression of the three PKC isoforms (as demonstrated by immunoblot analysis) and was accompanied by a redistribution of cPKC alpha and nPKC epsilon toward the particulate fraction. In TSH-treated cells, hydroxyapatite chromatography of cytosolic PKC revealed an additional peak of PKC activity eluted at 195 mM potassium phosphate. Its elution molarity did not correspond to the molarity of any known PKC isozyme, and it did not cross-react with antibodies directed against cPKC isozymes--: alpha, beta, or gamma. When TSH was replaced by forskolin (10(-5) M), identical quantitative and qualitative modifications were obtained, suggesting that, in thyroid cells, the cyclic AMP-dependent regulatory cascade could be involved in the control of PKC isoforms expression by TSH.

TSH action on cAMP binding to the regulatory subunits of cAMP-dependent protein kinases in pig thyroid cell cultures.

This study examines the mechanism of TSH action on the cAMP-dependent protein kinases (PKA) by measuring the catalytic activity of the two PKA isozymes (PKA I and PKA II) and their capacity to bind cAMP to the regulatory subunits (RI and RII) in thyroid cell cultures exposed for two days to different doses of TSH. In TSH-treated cell cultures a selective down regulation (up to 60%) of the catalytic activity was found; the PKA I was down regulated at lower TSH doses (0.1 mU/ml and even 0.05 mU/ml) than was the PKA II (1.0 mU/ml TSH). At the dose of 1.0 mU/ml the loss of the catalytic activity in PKA I and PKA II was respectively 60% and 40%. No free catalytic activity was found either in control or in TSH-treated cells. Binding of cAMP to regulatory subunits (R) measured under exchange conditions at 37 degrees C, showed that no change in total regulatory subunit protein content occurs in TSH-treated cells. Binding of cAMP to R subunits at 4 degrees C (when only free cAMP binding sites are measured) revealed an important endogenous occupancy of cAMP binding sites of RI and RII isoreceptors under basal conditions (40%) and a significantly increased occupancy after exposure of cells to TSH (60%). Pools of regulatory subunits with more than 50% of sites occupied, which were devoid of enzyme activity, were found both, in control and TSH-exposed cells. They were identified as RI subunits which represented a mixed population of native and partly degraded molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Species differences of the thyroid protein kinase C heterogeneity.

Protein kinase C (PKC), the mediator of the phosphoinositide transduction pathway, is a family of at least 11 isozymes and its heterogeneity has been described in many tissues and cells. We studied here the heterogeneity of PKC in thyroid glands from three different species, rat, pig, and dog. By combining immunological and biochemical approaches, we identified in rat thyroids, the PKC alpha, beta II, delta, epsilon, and zeta subspecies, in pig thyroids, the alpha, epsilon, and zeta isozymes, and in dog thyroids, only the alpha and zeta isozymes. The observed species differences of the thyroid gland PKC heterogeneity could be related to the reported species differences in the activation of the phosphoinositide regulatory cascade by TSH and other thyroid cell regulators.

Immunological identification of protein kinase C-alpha and protein kinase C-delta in cultured rat mesangial cells: differential sensitivity of the two isoforms towards the protein kinase inhibitor H7.

Rat mesangial cells contain both calcium-dependent protein kinase C (PKC) activity, which phosphorylates histone H1 and endogenous proteins, and calcium-independent, phospholipid-dependent PKC activity, which phosphorylates only endogenous proteins. The calcium-dependent PKC was identified as PKC alpha by immunoblot analysis and hydroxyapatite chromatography (HPLC). The calcium-insensitive, phospholipid-dependent isoform was identified as PKC delta using similar techniques. The inhibition of these two PKC isoforms by the protein kinase inhibitor H7 [1-(iso-quinolinyl sulphonyl)-2-methyl piperazine] was examined using both histone H1 and endogenous proteins as substrates. Phosphorylations catalyzed by the calcium-dependent PKC isoform alpha were almost 90% inhibited when histone H1 was used, and only 55% when endogenous proteins were the substrate. In contrast, the phosphorylation of endogenous proteins catalysed by the calcium-insensitive, phospholipid-dependent PKC delta was not significantly affected by the inhibitor.

Heterogeneity of protein kinase C in cultured rat mesangial cells.

Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity. Upon hydroxyapatite chromatography (HPLC), the PKC activity was eluted as a main peak at 150 mM potassium phosphate with a shoulder at 180 mM. Both peaks corresponded to the type III PKC from rat brain and were identified as PKC alpha isoform by immunoblot analysis. In contrast with what was observed using histone H1, the increased phosphorylation of endogenous proteins in the presence of a mixture of Ca2+/PS, plus either DAG or PMA, was only partly reduced by EGTA. Moreover, the level of the PKC activity detected in the presence of EGTA was comparable to the level of kinase activity, measured in the presence of PS alone or associated with DAG or PMA. This suggests that mesangial cells contain PKC activity which does not absolutely require calcium. Polyacrylamide gel electrophoresis revealed that patterns of phosphorylated mesangial cell proteins are different depending on whether calcium was added or not. In the presence of calcium, PKC strongly phosphorylated the proteins of 53,000 molecular weight, a doublet of 37,000-39,000, the 24,000 and the triplet of 17,000-20,000-22,000 molecular weight. The addition of EGTA to the assays suppressed completely the labelling of most proteins; only the 20,000 molecular weight protein remained strongly labelled, while the 39,000 molecular weight band was only faintly visible. The same patterns of phosphorylations were obtained after omission of calcium in the assays containing only PS and DAG (or PMA). So, the main substrates of calcium-dependent PKC are proteins of 53,000, 39,000, 37,000, 22,000, 24,000 and 17,000 molecular weight while the protein of 20,000 molecular weight appears to be the main substrate of calcium-independent PKC. The existence in mesangial cells of at least two forms of PKC, which phosphorylate specific endogenous proteins, emphasizes the complexity of the phospholipid-dependent regulatory cascade and raises the possibility that actions of different regulators may be transduced through distinct PKC isozymes.