Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation

The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation.It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.     

原代培养过程中壳聚糖衍生物对CTCF/YB-1/C-myc和p53蛋白表达的影响

成功建立了人增生性瘢痕细胞和正常皮肤成纤维细胞的原代培养, 并利用热休克蛋白(HSP47)和成纤维细胞特异蛋白(FSP)标记物进行了鉴定。研究发现, 经过壳聚糖衍生物处理, 人增生性瘢痕成纤维细胞和正常皮肤成纤维细胞在培养中均出现了不同类型的蛋白表达。多功能转录因子蛋白(CTCF)在壳聚糖衍生物处理的增生性瘢痕成纤维细胞中出现表达上调; 在聚糖衍生物处理的正常皮肤成纤维细胞中数量无变化。YB-1结合蛋白在经壳聚糖处理的正常皮肤成纤维细胞与人增生性瘢痕细胞中的表达几乎无异, 但在未经壳聚糖处理的细胞中表达不同。C-MYC和P53蛋白在壳聚糖衍生物处理的增生性瘢痕纤维细胞中表达上调, 但在正常皮肤成纤维细胞中, 无论是否经过壳聚糖衍生物处理, 这两种蛋白都没有表达。上述4种蛋白在人增生性瘢痕细胞和正常皮肤成纤维细胞中表现出不同的表达方式, 这种新型壳聚糖衍生物可能在控制人增生性瘢痕细胞和正常皮肤成纤维细胞生长和增殖过程中起着重要作用。这些蛋白因子的表达机制目前还不是完全清楚, 有待于进一步研究。     

Deletion of the PDGFR-beta gene affects key fibroblast functions important for wound healing

This study provides new perspectives of the unique aspects of platelet-derived growth factor beta-receptor (PDGFR-beta) signaling and biological responses through the establishment of a mutant mouse strain in which two loxP sequences were inserted into the introns of PDGFR-beta genome sequences. Isolation of skin fibroblasts from the mutant mice and Cre recombinase transfection in vitro induced PDGFR-beta gene deletion (PDGFR-betaDelta/Delta). The resultant depletion of the PDGFR-beta protein significantly attenuated platelet-derived growth factor (PDGF)-BB-induced cell migration, proliferation, and protection from H2O2-induced apoptosis of the cultured PDGFR-betaDelta/Delta dermal fibroblasts. PDGF-AA and fetal bovine serum were mitogenic and anti-apoptotic but were unable to induce the migration in PDGFR-beta Delta/Delta fibroblasts. Concerning the PDGF signaling, PDGF-BB-induced phosphorylation of Akt, ERK1/2, and JNK, but not p38, decreased in PDGFR-betaDelta/Delta fibroblasts, but PDGF-AA-induced signaling was not altered. Overexpression of the phospholipid phosphatases, SHIP2 and/or PTEN, inhibited PDGF-BB-induced phosphorylation of Akt and ERK1/2 in PDGFR-betaDelta/Delta fibroblasts but did not affect that of JNK and p38. These results indicate that disruption of distinct PDGFR-beta signaling pathways in PDGFR-betaDelta/Delta dermal fibroblasts impaired their proliferation and survival, but completely inhibits migratory response, and that PDGF-BB-induced phosphorylation of Akt and ERK1/2 possibly mediated by PDGFR-alpha is regulated, at least in part, by the lipid phosphatases SHIP2 and/or PTEN. Thus, the PDGFR-beta function on dermal fibroblasts appears to be critical in PDGF-BB action for skin wound healing and is clearly distinctive from that of PDGFR-alpha in the ligand-induced biological responses and the underlying properties of cellular signaling.     

Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts

Ultraviolet (UV) irradiation regulates UV-responsive genes, including matrix metalloproteinases (MMPs). Moreover, UV-induced MMPs cause connective tissue damage and the skin to become wrinkled and aged. Here, we investigated the effect of eicosapentaenoic acid (EPA), a dietary omega-3 fatty acid, on UV-induced MMP-1 expression in human dermal fibroblasts (HDFs). We found that UV radiation increases MMP-1 expression and that this is mediated by p44 and p42 MAP kinase (ERK) and Jun-N-terminal kinase (JNK) activation but not by p38 activation. Pretreatment of HDFs with EPA inhibited UV-induced MMP-1 expression in a dose-dependent manner and also inhibited the UV-induced activation of ERK and JNK by inhibiting ERK kinase (MEK1) and SAPK/ERK kinase 1 (SEK1) activation, respectively. Moreover, inhibition of ERK and JNK by EPA resulted in the decrease of c-Fos expression and c-Jun phosphorylation/expression induced by UV, respectively, which led to the inhibition of UV-induced activator protein-1 DNA binding activity. This inhibitory effect of EPA on MMP-1 was not mediated by an antioxidant effect. We also found that EPA inhibited 12-O-tetradecanoylphorbol-13-acetate- or tumor necrosis factor-alpha-induced MMP-1 expression in HDFs and UV-induced MMP-1 expression in HaCaT cells. In conclusion, our results demonstrate that EPA can inhibit UV-induced MMP-1 expression by inhibiting the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways. Therefore, EPA is a potential agent for the prevention and treatment of skin aging.     

Opposing effects of protein kinase Calpha and protein kinase Cepsilon on collagen expression by human lung fibroblasts are mediated via MEK/ERK and caveolin-1 signaling

The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.     

Protein kinase Czeta attenuates hypoxia-induced proliferation of fibroblasts by regulating MAP kinase phosphatase-1 expression

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.     

The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-β1 concentrations during primary fibroblast proliferation

Increasing evidence has suggested that bidirectional regulation of cell proliferation is one important effect of TGF-β1 in wound healing. Increased c-Ski expression plays a role in promoting fibroblast proliferation at low TGF-β1 concentrations, but the mechanism by which low TGF-β1 concentrations regulate c-Ski levels remains unclear. In this study, the proliferation of rat primary fibroblasts was assessed with an ELISA BrdU kit. The mRNA and protein expression and phosphorylation levels of corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting. We first found that low TGF-β1 concentrations not only promoted c-ski mRNA and protein expression in rat primary fibroblasts but also increased the phosphorylation levels of Extracellular Signal-Regulated Kinases (ERK) and cAMP response element binding (CREB) protein. An ERK kinase (mitogen-activated protein kinase kinase, MEK) inhibitor significantly inhibited ERK1/2 phosphorylation levels, markedly reducing c-Ski expression and CREB phosphorylation levels and abrogating the growth-promoting effect of low TGF-β1 concentrations. At the same time, Smad2/3 phosphorylation levels were not significantly changed. Taken together, these results suggest that the increased cell proliferation induced by low TGF-β1 concentrations mediates c-Ski expression potentially through the ERK/CREB pathway rather than through the classic TGF-β1/Smad pathway.     

Induction of hepatocyte growth factor expression by maleic acid in human fibroblasts through MAPK activation

Carboxylic acids have various biological activities and play critical roles in cellular metabolic pathways such as the tricarboxylic acid (TCA) cycle. It has been shown that some carboxylic acids induce cell proliferation and production of cytokines or growth factors. However, there have been no reports on effects of carboxylic acids on hepatocyte growth factor (HGF) expression. In this study, we found that only maleic acid among various carboxylic acids examined markedly induced HGF production from human dermal fibroblasts. Maleic acid also induced HGF production from human lung fibroblasts and neuroblastoma cells. The stimulatory effect was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) but not in phosphorylation of p38 was observed from 6 h and up to 24 h after maleic acid addition. The ERK kinase inhibitor PD98059 and the JNK inhibitor SP600125 potently inhibited maleic acid-induced HGF production, while the p38 inhibitor SB203580 did not significantly inhibit the production. The protein synthesis inhibitor cycloheximide completely inhibited upregulation of HGF mRNA induced by maleic acid but superinduced HGF mRNA expression upregulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These results suggest that maleic acid indirectly induced HGF expression from human dermal fibroblasts through activation of ERK and JNK and that de novo protein synthesis is required for maleic acid-induced upregulation of HGF mRNA.     

Activated Protein C Inhibits Proliferation and Tumor Necrosis Factor α–Stimulated Activation of p38, c-Jun NH2-Terminal Kinase (JNK) and Akt in Rheumatoid Synovial Fibroblasts

Synovial fibroblast proliferation is a hallmark of the invasive pannus in the rheumatoid joint. Activated protein C (APC) is a natural anticoagulant that exerts antiinflammatory and cyto-protective effects in various diseases via endothelial protein C receptor (EPCR) and proteinase-activated receptor (PAR)-mediated pathways. In this study, we investigated the effect and the underlying cellular signaling mechanisms of APC on proliferation of human rheumatoid synovial fibroblasts (RSFs). We found that APC stimulated proliferation of mouse dermal fibroblasts (MDFs) and normal human dermal fibroblasts (HDFs) by up to 60%, but robustly downregulated proliferation of RSFs. APC induced the phosphorylation of extracellular signal–regulated protein kinase (ERK) and enhanced expression of p21 and p27 in a dose-dependent manner in RSFs. The latter effect was inhibited by pre-treatment with the ERK inhibitors PD98059 and U0126 but not by p38 inhibitor SB203580. In addition, APC significantly downregulated tumor necrosis factor (TNF)α-stimulated cell proliferation and activation of p38, c-Jun NH₂-terminal kinase (JNK) and Akt in RSFs. These results provide the first evidence that APC selectively inhibits proliferation and the inflammatory signaling pathways of RSFs. Thus, APC may reduce synovial hyperplasia and pannus invasion in rheumatoid arthritis.     

Repetitive deformation activates focal adhesion kinase and ERK mitogenic signals in human Caco-2 intestinal epithelial cells through Src and Rac1

Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown. We investigated whether Src and Rac1 mediate deformation-induced FAK and ERK phosphorylation and proliferation in human Caco-2 and rat IEC-6 intestinal epithelial cells. Cells cultured on collagen-I were subjected to an average 10% cyclic strain at 10 cycles/min. Cyclic strain activated Rac1 and induced Rac1 translocation to cell membranes. Mechanical strain also induced rapid sustained phosphorylation of c-Src at Tyr(418), Rac1 at Ser(71), FAK at Tyr(397) and Tyr(576), and ERK1/2 at Thr(202)/Tyr(204). The mitogenic effect of cyclic strain was blocked by inhibition of Src (PP2 or short interfering RNA) or Rac1 (NSC23766). Src or Rac1 inhibition also prevented strain-induced FAK phosphorylation at Tyr(576) and ERK phosphorylation but not FAK phosphorylation at Tyr(397). Reducing FAK using short interfering RNA blocked strain-induced mitogenicity and attenuated ERK phosphorylation but not Src or Rac1 phosphorylation. Src inhibition blocked strain-induced Rac1 phosphorylation, but Rac inhibition did not alter Src phosphorylation. Transfection of a two-tyrosine phosphorylation-deficient FAK mutant Y576F/Y577F prevented activation of cotransfected myc-ERK2 by cyclic strain. Repetitive deformation induced by peristalsis or villus motility may support the gut mucosa by a pathway involving Src, Rac1, FAK, and ERK. This pathway may present important targets for interventions to prevent mucosal atrophy during prolonged ileus or fasting.     

PED interacts with Rac1 and regulates cell migration/invasion processes in human non‐small cell lung cancer cells

PED (phosphoprotein enriched in diabetes) is a 15 kDa protein involved in many cellular pathways and human diseases including type II diabetes and cancer. We recently reported that PED is overexpressed in human cancers and mediates resistance to induced apoptosis. To better understand its role in cancer, we investigated on PED interactome in non‐small cell lung cancer (NSCLC). By the Tandem Affinity Purification (TAP), we identified and characterized among others, Rac1, a member of mammalian Rho GTPase protein family, as PED‐interacting protein. In this study we show that PED coadiuvates Rac1 activation by regulating AKT mediated Rac1‐Ser⁷¹ phosphorylation. Furthermore, we show that the expression of a constitutively active Rac, affected PED‐Ser¹⁰⁴ phosphorylation, which is important for PED‐regulated ERK 1/2 nuclear localization. Through specific Rac1‐siRNA or its pharmacological inhibition, we demonstrate that PED augments migration and invasion in a Rac1‐dependent manner in NSCLC. In conclusion, we show for the first time that PED and Rac1 interact and that this interaction modulates cell migration/invasion processes in cancer cells through ERK1/2 pathway. J. Cell. Physiol. 225: 63–72, 2010. © 2010 Wiley‐Liss, Inc.     

TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation; interestingly its action is clearly cell type-dependent. In particular, it inhibits epithelial and endothelial cells' proliferation, while its action on many mesenchymal cells has been reported to be stimulatory. In this direction, we have recently shown that TGF-beta regulates the proliferation of normal human skin fibroblasts according to their developmental origin: i.e. it inhibits fetal fibroblasts, while it stimulates the proliferation of adult ones. Here, we present evidence on the mechanisms underlying this differential action. Concerning fetal fibroblasts, we have found that TGF-beta activates Protein Kinase A (PKA) and induces the expression of the cyclin-dependent kinase inhibitors (CKIs) p21(CIP1/WAF1) and p15(INK4B). Moreover, the specific PKA inhibitor H-89 blocks the induction of both CKIs and annuls the TGF-beta-mediated inhibitory effect, indicating the central role of PKA in this process. In contrast, in adult cells no PKA activation is observed. Moreover, TGF-beta stimulates cell proliferation by activating the MEK-ERK pathway, as the MEK inhibitor PD98059 blocks this effect. A specific neutralizing antibody against Fibroblast Growth Factor-2 (FGF-2) inhibits both ERK activation and the mitogenic activity of TGF-beta, indicating that the latter establishes an autocrine loop, via FGF-2, leading to cell proliferation. This loop requires FGF receptor-1 (FGFR-1), as its down-regulation by siRNA approach prevents TGF-beta from stimulating ERK-1/2 activation and DNA synthesis. In conclusion, the differential proliferative response of fetal and adult normal human skin fibroblasts to TGF-beta is regulated by distinct signaling pathways and furthermore it may provide information on the bimodal effect of this factor on cell proliferation, in general.     

Despite activation of EGF-receptor-ERK signaling pathway, epithelial proliferation is impaired in portal hypertensive gastric mucosa: relevance of MKP-1, c-fos, c-myc, and cyclin D1 expression.

Portal hypertensive (PHT) gastric mucosa has increased susceptibility to injury and impaired mucosal healing. Our previous study demonstrated increased ERK activation and MAP kinase phosphatase-1 (MKP-1) overexpression in PHT gastric mucosa. However, it remains unknown which tyrosine kinase receptors are involved in ERK activation and whether ERK activation results in increased cell proliferation. We examined whether EGF receptor (EGF-R) is involved in ERK activation and whether ERK activation triggers epithelial proliferation in PHT gastric mucosa. In gastric mucosa of PHT and sham-operated (SO) rats we studied: (1) EGF-R mRNA and protein expression as well as phosphorylation and membrane protein tyrosine kinase (PTK) activity; (2) ERK2 phosphorylation and activity; (3) MKP-1 mRNA and protein; (4) c-fos, c-myc and cyclin D1 mRNAs, and gastric epithelial proliferation. In PHT gastric mucosa: (1) EGF-R mRNA, protein and phosphorylation and membrane PTK activity were all significantly increased by 38%, 49%, 43% and 49%, respectively; (2) ERK2 phosphorylation and activity were significantly increased by 40% and 50 %, respectively; (3) MKP-1 mRNA and protein expression were significantly increased by 27% and 34%, respectively. In contrast, (4) c-fos, c-myc, and cyclin D1 mRNAs expression were all significantly decreased in PHT gastric mucosa by 36%, 33%, and 49%, respectively, and cell proliferation was significantly lower that in SO rats (11% in PHT vs. 18% in SO). These results suggest that in PHT gastric mucosa, ERK activation is mediated through EGF-R upregulation, but the gastric epithelial proliferation is impaired, possibly by MKP-1 overexpression, leading to reduction of c-fos, c-myc and cyclin D1.     

皮肤血管瘤组织中WT-1、Bcl-2、P53的表达及意义

目的研究肾母细胞瘤基因（WT-1）、Bcl-2和P53在增生期、退化期血管瘤及正常组织中的表达,探讨其意义及相互关联。方法采用免疫组化SP法检测人皮肤血管瘤组织中WT1、Bcl-2和P53在增生期、退化期及正常皮肤组织中血管内皮细胞中的表达水平,利用计算机成像分析技术检测不同时期皮肤血管瘤与正常皮肤组织WT1、Bcl-2和P53的平均光密度及其阳性面积率。结果1.WT-1在退化期血管瘤中有较强表达,而在增生期血管瘤和正常皮肤组织中表达微弱或不表达（P〈0.05）。2.Bcl-2在增生期血管瘤的表达明显高于退化期血管瘤和正常皮肤组织（P〈0.01）;Bcl-2在退化期血管瘤的表达与正常皮肤组织相比,差异无显著性（P〉0.05）。3.p53基因在增生期血管瘤组织中表达水平高于退化期,差异有极显著性意义（P〈0.01）,退化期血管瘤p53基因表达水平与正常皮肤组织相比,差异无显著性意义（P〉0.05）。结论1.WT-1可能通过促进内皮细胞凋亡而抑制血管瘤的增生;2.Bcl-2可能是通过抑制内皮细胞的凋亡,使其增殖和凋亡失衡;3.P53可能促进了血管瘤增生期内皮细胞的增殖,使血管内皮细胞大量生成。     

MAP kinase mediates silica-induced fibrotic nodule formation and collagen accumulation in fibroblasts

It is well known that silica generates fibrosis around them in animals and human. However, the pathogenesis and mechanism of silica-induced fibrosis are still poorly understood. Here, we established a new strategy through which the effects of silica on fibrotic nodule formation, key extracellular matrix accumulation, and the mechanism involved were explored. To achieve this, human dermal fibroblasts were directly exposed to silica gel for various durations. Fibrotic nodule formation was evaluated by their microscopic appearance, type-1 procollagen, and fibronection expression in cell lysate and MMP-1 and-3 in conditioned media were analyzed by Western blotting. The results show an easily formation of nodule-like structures around silica gel in an in vitro-cultured system. The findings further revealed that silica gel stimulates collagen and fibronectin expression, while down-regulates matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) released in conditioned medium. To explore the mechanism involved, P38 and ERK1/2 Mitogen-Activated Protein Kinase (MAPK) signaling pathways were evaluated. Result showed that silica inhibits P38 and extracellular signal-regulated kinases (ERK1/2) MAP kinase phosphorylation. The addition of ERK1/2 inhibitor increases silica-stimulated type-1 collagen expression, reduces MMP-1 release and further enhances silica-induced nodule formation in dermal fibroblasts. These findings indicate that the inhibition of ERK1/2 MAPK signaling pathway may contribute to silica-caused fibrosis. In summary, our findings suggest that silica can directly cause fibrotic phenotype when fibroblasts contact with silica particles independent of any inflammation and other factors may exist in an in vivo condition.     

Dependence of corneal epithelial cell proliferation on modulation of interactions between ERK1/2 and NKCC1

Epidermal growth factor (EGF) receptor stimulation or protein kinase C (PKC) activation enhances corneal epithelial cell proliferation. This response is needed to maintain corneal transparency and vision. We clarify here in human corneal epithelial cells (HCEC) the cause and effect relationships between ERK1/2 and NKCC1 phosphorylation induced by EGF receptor or PKC activation. Furthermore, the roles are evaluated of NF-κB and ERK1/2 in mediating negative feedback control of ERK1/2 and NKCC1 phosphorylation through modulating DUSP1 and DUSP6 expression levels. Intracellular Ca(2+) rises induced by EGF elicited NKCC1 phosphorylation through ERK1/2 activation. Bumetanide suppressed EGF-induced NKCC1 phosphorylation, transient cell swelling and cell proliferation. This cause and effect relationship is similar to that induced by PKC stimulation. NKCC1 activation occurred through time-dependent increases in protein-protein interaction between ERK1/2 and NKCC1, which were proportional to EGF concentration. DUSP6 upregulation obviated EGF and PKC-induced NKCC1 phosphorylation. NF-κB inhibition by PDTC prolonged ERK1/2 activation through GSK-3 inactivation leading to declines in DUSP1 expression levels. These results show that EGF receptor and PKC activation induce increases in HCEC proliferation through ERK1/2 interaction with NKCC1. This response is modulated by changes in DUSP1- and DUSP6-mediated negative feedback control of ERK1/2-induced NKCC1 phosphorylation.