Protein Kinase C Attenuates Hypoxia-induced Proliferation of Fibroblasts by Regulating MAP Kinase Phosphatase-1 Expression

Developmental Lung Biology Research Laboratory, Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 05/2006; 17(4):1995-2008. DOI: 10.1091/mbc.E05-09-0869
Source: PubMed


We have previously found that hypoxia stimulates proliferation of vascular fibroblasts through Galphai-mediated activation of ERK1/2. Here, we demonstrate that hypoxia also activates the atypical protein kinase Czeta (PKCzeta) isozyme and stimulates the expression of ERK1/2-specific phosphatase, MAP kinase phosphatase-1 (MKP-1), which attenuates ERK1/2-mediated proliferative signals. Replication repressor activity is unique to PKCzeta because the blockade of classical and novel PKC isozymes does not affect fibroblast proliferation. PKCzeta is phosphorylated upon prolonged (24 h) exposure to hypoxia, whereas ERK1/2, the downstream kinases, are maximally activated in fibroblasts exposed to acute (10 min) hypoxia. However, PKCzeta blockade results in persistent ERK1/2 phosphorylation and marked increase in hypoxia-induced replication. Similarly prolonged ERK1/2 phosphorylation and increase in hypoxia-stimulated proliferation are also observed upon blockade of MKP-1 activation. Because of the parallel suppressive actions of PKCzeta and MKP-1 on ERK1/2 phosphorylation and proliferation, the role of PKCzeta in the regulation of MKP-1 expression was evaluated. PKCzeta attenuation reduces MKP-1 expression, whereas PKCzeta overexpression increases MKP-1 levels. In conclusion, our results indicate for the first time that hypoxia activates PKCzeta, which acts as a terminator of ERK1/2 activation through the regulation of downstream target, MKP-1 expression and thus serves to limit hypoxia-induced proliferation of fibroblasts.

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Available from: Kurt Stenmark, Mar 10, 2014
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    • "Das et al. found that bovine pulmonary artery adventitial fibroblasts expressed cPKCα, βI and βII, nPKCδ and ɛ, and aPKCζ, but only cPKCβI and aPKCζ activations were associated with the exaggerated growth responses of pulmonary artery adventitial fibroblasts under chronic hypoxia condition [16,22]. However, Short et al. found that aPKCζ attenuates hypoxia-induced proliferation of fibroblasts by regulating MAP kinase phosphatase-1 (MKP-1) expression [23]. "
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    ABSTRACT: Evidence indicates that protein kinase C (PKC) plays a pivotal role in hypoxia-induced pulmonary hypertension (PH), but PKC isoform-specific protein expression in pulmonary arteries and their involvement in hypoxia-induced PH are unclear. Male SD rats (200-250 g) were exposed to normobaric hypoxia (10% oxygen) for 1, 3, 7, 14 and 21 d (days) to induce PH. PKC isoform-specific membrane translocation and protein expression in pulmonary arteries were determined by using Western blot and immunostaining. We found that only 6 isoforms of conventional PKC (cPKC) α, βI and βII, and novel PKC (nPKC) δ, ε and η were detected in pulmonary arteries of rats by Western blot. Hypoxic exposure (1-21 d) could induce rat PH with right ventricle (RV) hypertrophy and vascular remodeling. The cPKCβII membrane translocation at 3-7 d and protein levels of cPKCα at 3-14 d, βI and βII at 1-21 d decreased, while the nPKCδ membrane translocation at 3-21 d and protein levels at 3-14 d after hypoxic exposure in pulmonary arteries increased significantly when compared with that of the normoxia control group (p<0.05 vs. 0 d, n=6 per group). In addition, the down-regulation of cPKCα,βI and βII, and up-regulation of nPKCδ protein expressions at 14 d after hypoxia were further confirmed by immunostaining. This study is the first systematic analysis of PKC isoform-specific membrane translocation and protein expression in pulmonary arteries, suggesting that the changes in membrane translocation and protein expression of cPKCα, βI, βII and nPKCδ are involved in the development of hypoxia-induced rat PH.
    Preview · Article · Feb 2012 · Medical science monitor: international medical journal of experimental and clinical research
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    • "After 1 h, cell fractions were analyzed by western analyses. PKCz has been implicated in ERK1/2 activation and was therefore selected as the focused isotype (Short et al. 2006). The results shown in Fig. 3B indicate activated PKCz in membrane fractions (PKCz (M), row 1) whereas the inactivated forms are detected in the cytosolic fraction (PKCz (C), row 2). "
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    ABSTRACT: The chemokine Stromal-derived factor-1alpha (SDF-1alpha) interacts with seven transmembrane (TM) G-protein-coupled receptor (GPR), CXCR4. SDF-1alpha is linked to inflammation, chemoattraction, cancer metastasis, and hematopoiesis. Tachykinin (Tac1) peptides bind seven transmembrane (TM), GPR and are involved in tumor promotion. SDF-1alpha regulates Tac1 expression in non-tumorigenic breast cells through a bimodal pattern with repression at high levels through nuclear factor-kappa B (NFkappaB) activation. This study focuses on the mechanism of activation at low SDF-1alpha in MCF12A non-tumorigenic breast cells. Reporter gene assays with the 5' flanking region of Tac1 (exon 1 omitted) and co-transfection with the repressor of cAMP response element (CREB) (ICER), and transfection with the CRE sites mutated, verified critical roles for CRE sites in SDF-1alpha-mediated Tac1 activation. Western blots and functional assays with specific inhibitors indicated that SDF-1alpha phosphorylated CREB (P-CREB) via Galpha(i)2-PI3K-protein kinase C (PKC)zeta-p38-extracellular signal-regulated kinase (ERK) and no evidence of cAMP-PKA pathway. This observation is different from previous studies that reported CREB-phosphorylated PKA pathway in the activation of Tac1 in bone marrow stromal cells. This suggests cell specificity in Tac1 expression. In conclusion, this study reports on a non-canonical pathway in Tac1 activation by SDF-1alpha. This finding is significant, since Tac1 is relevant to breast cancer metastasis, to bone marrow where stromal cells have a significant facilitating function.
    Preview · Article · Apr 2008 · Journal of Molecular Endocrinology
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    • "TGF-β signaling and retinoid acid signaling upregulate MKP-1 expression [53] [54]. Moreover, while PKCζ induces MKP-1 expression in response to hypoxia [55], PKCδ triggers MKP-1 degradation in glutamate-induced cell death [56]. In addition, MKP-1 is induced by several hormones, such as glucocorticoids , endocannabinoid and parathyroid [57] [58] [59]. "
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    ABSTRACT: MAP kinase phosphatases (MKPs) catalyze dephosphorylation of activated MAP kinase (MAPK) molecules and deactivate them. Therefore, MKPs play an important role in determining the magnitude and duration of MAPK activities. MKPs constitute a structurally distinct family of dual-specificity phosphatases. The MKP family members share the sequence homology and the preference for MAPK molecules, but they are different in substrate specificity among MAPK molecules, tissue distribution, subcellular localization and inducibility by extracellular stimuli. Our understanding of their protein structure, substrate recognition mechanisms, and regulatory mechanisms of the enzymatic activity has greatly increased over the past few years. Furthermore, although there are a number of MKPs, that have similar substrate specificities, non-redundant roles of MKPs have begun to be identified. Here we focus on recent findings regarding regulation and function of the MKP family members as physiological regulators of MAPK signaling.
    Full-text · Article · Sep 2007 · Biochimica et Biophysica Acta
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