17β-estradiol inhibits TGF-β-induced collagen gel contraction mediated by human Tenon fibroblasts via Smads and MAPK signaling pathways.

Excessive wound contraction can lead to scar formation in the conjunctiva. The effects of all-trans-retinoic acid (ATRA) on the contractility of human Tenon fibroblasts (HTFs) cultured in three-dimensional (3D) collagen gels were investigated. Human Tenon fibroblasts were cultured in 3D gels of type I collagen and in the absence or presence of TGF-β, ATRA, or various inhibitors. Collagen gel contraction was evaluated by measurement of gel diameter. Phosphorylation of various signaling molecules was examined by immunoblot analysis. The formation of actin stress fibers and focal adhesions was detected by laser confocal microscopy. All-trans-retinoic acid inhibited TGF-β-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. The TGF-β-induced phosphorylation of focal adhesion kinase (FAK) and formation of stress fibers and focal adhesions in HTFs were attenuated by ATRA. All-trans-retinoic acid also inhibited the TGF-β-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) as well as that of c-Jun and Smad2/3. Furthermore, TGF-β-induced collagen gel contraction was blocked by inhibitors of ERK, p38, or JNK signaling. All-trans-retinoic acid inhibited TGF-β-induced collagen gel contraction mediated by HTFs, most likely by attenuating the formation of actin stress fibers and focal adhesions as well as signaling by MAPKs, c-Jun, and Smads. All-trans-retinoic acid may therefore prove effective for inhibition of conjunctival scarring through attenuation of the contractility of Tenon fibroblasts.

Collagenase-3 (MMP-13) Enhances Remodeling of Three-Dimensional Collagen and Promotes Survival of Human Skin Fibroblasts

To evaluate the effects of TGF-beta1 and doxycycline on production of gelatinase MMP-9 and activation of Smad, c-Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways in human corneal epithelial cells. Primary human corneal epithelial cells were cultured to confluence. The cells were treated with different concentrations of TGF-beta1 (0.1, 1, or 10 ng/mL), with or without TGF-beta1-neutralizing mAb (5 microg/mL), SP600125 (30 microM), PD98059 (40 microM), SB202190 (20 microM), or doxycycline (5-40 microg/mL) for different lengths of time. Conditioned media were collected from cultures treated for 24 to 48 hours to evaluate the MMP-9 production by zymography and activity assay. Total RNA was isolated from cells treated for 6 to 24 hours to evaluate MMP-9 expression by semiquantitative RT-PCR and Northern hybridization. Cells treated for 5 to 60 minutes were lysed in RIPA buffer for Western blot with phospho-specific antibodies against Smad2, JNK1/2, ERK1/2, or p38. TGF-beta1 increased expression, production, and activity of MMP-9 by human corneal epithelial cells in a concentration-dependent fashion. TGF-beta1 also induced activation of Smad2, JNK1/2, ERK1/2, and p38 within 5 to 15 minutes, with peak activation at 15 to 60 minutes. Doxycycline markedly inhibited the TGF-beta1-induced production of MMP-9 and activation of the Smad, JNK1/2, ERK1/2, and p38 signaling pathways. Its inhibitory effects were of a magnitude similar to SP600125, PD98059, and SB202190, specific inhibitors of the JNK1/2, ERK1/2, and p38 pathways, respectively. These findings demonstrated that doxycycline inhibits TGF-beta1-induced MMP-9 production and activity, perhaps through the Smad and MAPK signaling pathways. These inhibitory effects may explain the reported efficacy of doxycycline in treating MMP-9-mediated ocular surface diseases.

Background/aimsScarring and contraction of the conjunctiva are common complications of many ocular diseases. We investigated the effects of all-trans-retinoic acid (ATRA) on the contractility of human Tenon's capsule fibroblasts (HTFs)...

The mitogen-activated protein kinase (MAPK) signaling pathway, particularly the p38 MAPK and ERK1/2, has been implicated in the pathogenesis of Parkinson's disease (PD). Recent studies have shown that MAPK signaling pathway can influence the expression of matrix metalloproteinase 9 (MMP-9), known for its involvement in various physiological and pathological processes, including neurodegenerative diseases. This study explores the modulation of MMP-9 expression via the MAPK/ERK signaling cascade and its potential therapeutic implications in the context of PD-associated motor dysfunction. Here, tolperisone hydrochloride (TL), a muscle relaxant that blocks voltage-gated sodium and calcium channels, was used as a treatment to observe its effect on MAPK signaling and MMP-9 expression. Rotenone (RT) exposure in mice resulted in a significant reduction in substantia nigra and primary motor cortex neurons, which were further evidenced by impairments in motor function. When TL was administered, neuron count was restored (89.0 ± 4.78 vs 117.0 ± 4.46/mm2), and most of the motor dysfunction was alleviated. Mechanistically, TL reduced the protein expression of phospho-p38MAPK (1.06 fold vs 1.00 fold) and phospho-ERK1/2 (1.16 fold vs 1.02 fold), leading to the inhibition of MAPK signaling, as well as reduced MMP-9 concentrations (2.76 ± 0.10 vs 1.94 ± 0.10 ng/mL) in the process of rescuing RT-induced neuronal cell death and motor dysfunction. Computational analysis further revealed TL’s potential inhibitory properties against MMP-9 along with N and L-type calcium channels. These findings shed light on TL's neuroprotective effects via MMP-9 inhibition and MAPK signaling downregulation, offering potential therapeutic avenues for PD-associated motor dysfunction.

Subconjunctival fibrosis represents the primary cause of postoperative failure of trabeculectomy, and at present there is a lack of effective intervention strategies. The present study aimed to investigate the effect of the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 on human tenon fibroblast (HTF) myofibrosis transdifferentiation, and to illuminate the underlying molecular mechanisms involved. It was demonstrated that U0126 significantly inhibited the proliferation, migration and collagen contraction of HTFs stimulated with TGF-β1. In addition, U0126 largely attenuated the TGF-β1-induced conversion of HTFs into myofibroblasts, as indicated by a downregulation of the mRNA and protein expression of α-smooth muscle actin and zinc finger protein SNAI1, and by ameliorating the 3D-collagen contraction response. Mechanistically, U0126 suppressed the TGF-β1-stimulated phosphorylation of mothers against decapentaplegic homolog 2/3, P38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2, indicating that U0126 may inhibit HTF activation through the canonical and non-canonical signaling pathways of TGF-β1. Therefore, U0126 exhibits a potent anti-fibrotic effect among HTFs, and the inhibition of MEK signaling may serve as an alternative intervention strategy for the treatment of trabeculectomy-associated fibrosis.

ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-β1 Autoinduction in Proximal Tubular Epithelial Cells

The outcome of glaucoma filtration surgery is affected by subconjunctival wound healing. The effects of the antiglaucoma drug latanoprost on the contractility of human Tenon fibroblasts (HTFs) cultured in a three-dimensional collagen gel were investigated. HTFs were cultured in a type I collagen gel with latanoprost or various inhibitors of intracellular signaling. Collagen gel contraction was evaluated by measurement of gel diameter, and collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. Phosphorylation of mitogen-activated protein kinases (MAPKs), focal adhesion kinase (FAK), and myosin light chain (MLC) was assessed by immunoblot analysis, and the formation of actin stress fibers was examined by laser confocal microscopy. HTF-mediated collagen gel contraction was stimulated by latanoprost in a concentration- and time-dependent manner. Latanoprost had no effect on collagen degradation by HTFs. Latanoprost induced phosphorylation of MAPKs (ERK, p38, and JNK) and FAK, as well as the formation of stress fibers in HTFs. Furthermore, latanoprost-induced collagen gel contraction was reduced by inhibitors of ERK (PD98059 and ERK inhibitor II), p38 (SB203580), JNK (JNK inhibitor II), Rho-associated kinase (Y27632), phospholipase C (U73122), and MLC kinase (ML-7). Latanoprost induced collagen gel contraction mediated by HTFs. This action of latanoprost appeared to depend on the formation of stress fibers and the activation of MAPKs, FAK, Rho-associated kinase, phospholipase C, and MLC kinase in HTFs. Latanoprost may therefore influence subconjunctival wound healing by affecting the contractility of Tenon fibroblasts.

Mucin hypersecretion is commonly observed in many inflammatory diseases of the respiratory tract. MUC5AC is generally recognized to be a major airway mucin because MUC5AC is highly expressed in the goblet cells of human airway epithelium. Moreover, it is regulated by various inflammatory cytokines. However, the mechanisms by which the interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha induce MUC5AC gene expression in normal nasal epithelial cells, and the signal molecules involved, especially in the downstream signaling of mitogen-activated protein (MAP) kinases, remain unclear. Here we show that pharmacologic or genetic inhibition of either ERK or p38 MAP kinase pathway abolished IL-1beta- and TNF-alpha-induced MUC5AC gene expression in normal human nasal epithelial cells. Our results also indicate that the activation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-response element-binding protein and cAMP-response element signaling cascades via ERK and p38 MAP kinases are crucial aspects of the intracellular mechanisms that mediate MUC5AC gene expression. Taken together, these studies give additional insights into the molecular mechanism of IL-1beta- and TNF-alpha-induced MUC5AC gene expression and enhance our understanding on mucin hypersecretion during inflammation.

Introduction: The Nef-M1 peptide competes effectively with the natural ligand of CXCR4, SDF-1α, to induce apoptosis and inhibit growth in colon and breast cancers. Its mechanistic role in tumor angiogenesis, a key step involved in tumor growth and metastasis, is unknown. In this study, we evaluated the antiangiogenic effect of Nef-M1 and examined its role in the AKT and Mitogen-Activated Protein Kinase (MAPK) signaling pathways in colon cancer. Experimental Design: We studied HT29 and SW480 colon cancer cells in vitro and tumor xenografts developed from HT29 cells were propagated in severe combined immunodeficient mice in vivo. The mice were treated intraperitoneally with the Nef-M1 peptide or sNef-M1 (scramble peptide as control) starting at the time of tumor implantation. Sections from tumors were evaluated for tumor angiogenesis, as measured by microvessel density (MVD) based on immunostaining of endothelial markers (CD31 and FVIII-RAg). MVD was determined by light microscopy in areas of invasive tumor containing the highest numbers of microvessels per area. Individual microvessel counts were made on a 200x field within the areas of most intense tumor neovascularization. Western blot analyses were performed on lysates of both colon cancer cell lines and HT-29 tumors to assess the effect of Nef-M1 on the AKT, and MAPK signaling pathways. Results: Immunostaining analyses indicated that control tumors had well established vascularity, but Nef-M1 treated tumors had poor vascularization. In addition, the average MVD was reduced in Nef-M1 treated tumors (n=5) compared to sNef-M1 control tumors (n=12) (p<0.05). In Nef-M1 treated tumors, the average microvessel size was significantly decreased from 2.28μm2 to 1.16μm2. Western blot analyses of lysates of colon cancer cells and tumors revealed that Nef-M1 effectively suppressed the activation of p38 and extracellular signal-regulated kinase (ERK) MAP kinases. AKT activation was also inhibited in Nef-M1 treated samples of colon cancer cells and tumor xenografts. This inhibition was accompanied by down-regulation of phosphorylated glycogen synthase kinase-3β (p-GSK-3β), a downstream target of p-AKT. Conclusions: Our data suggest that Nef-M1 inhibits tumor angiogenesis by attenuating AKT and MAPK signaling pathways and that, by targeting chemokine receptor CXCR4 mediated pathways, it may be a novel therapeutic agent for colon cancers. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251. Citation Format: Venkat R. Katkoori, Marc D. Basson, Upender Manne, Harvey L. Bumpers. Nef-M1, a peptide antagonist of CXCR4, inhibits tumor angiogenesis by attenuating AKT and mitogen-activated protein kinase signaling in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1027. doi:10.1158/1538-7445.AM2014-1027

Reactive oxygen species and nuclear factor-kappa B pathway mediate high glucose-induced Pax-2 gene expression in mouse embryonic mesenchymal epithelial cells and kidney explants

Pathogenesis of Photoaging in Human Dermal Fibroblasts

Angiotensin II induces the production of MMP-3 and MMP-13 through the MAPK signaling pathways via the AT1 receptor in osteoblasts

Cartilage degradation is an important in the pathogenesis of osteoarthritis (OA). Abnormal activation of the mitogen-activated protein kinase (MAPK) signaling pathway in chondrocytes promotes an inflammatory response, resulting in the release of chondral matrix-degrading enzymes that accelerate the degradation of cartilage. As a non-pharmaceutical and non-invasive physical therapy regimen, warm sparse-dense wave (WSDW) has been successfully used for the treatment of OA. However, it remains unclear whether WSDW inhibits cartilage degradation in OA through the MAPK signaling pathway. The present study investigated the effects of WSDW on papain-induced OA in rat knee joints. Papain-induced OA was established in rats, which were subsequently divided into a model group and three experimental groups that received a WSDW with the following ratios: WSDW=1:1, WSDW=1:2 and WSDW=2:1. After 12 weeks of treatment, cartilage degradation was evaluated by Mankin scoring of paraffin-embedded sections stained with hematoxylin and eosin. The changes in cartilage structure were observed by transmission electron microscopy, and the expressions of RAS, extracellular signal-regulated kinase (ERK), p38 and p53 were measured by reverse transcription-quantitative polymerase chain reaction and western blot analysis. WSDW was demonstrated to improve the arrangement of collagen fibers, inhibit the tidemark replication and delay cartilage degradation in papain-induced OA. The expressions of RAS, ERK, p38 and p53 in the WSDW (1:2) and (2:1) groups were significantly decreased when compared with the model group (P<0.01). Furthermore, amongst the WSDW groups, the inhibitory effects of the WSDW (1:2) group were typically greater than those of the WSDW (1:1) and (2:1) groups. The results indicate that WSDW may inhibit cartilage degradation in papain-induced OA in rat knee joints by regulating the MAPK signaling pathway.

In neuronal precursor cells, the magnitude and longevity of mitogen-activated protein (MAP) kinase cascade activation contribute to the nature of the cellular response, differentiation, or proliferation. However, the mechanisms by which neurotrophins promote prolonged MAP kinase signaling are not well understood. Here we defined the Rin GTPase as a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling and neuronal development. Rin is expressed exclusively in neurons and is activated by neurotrophin signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to nerve growth factor (NGF)-mediated neuronal differentiation. Most surprisingly, although Rin was unable to stimulate MAP kinase activity in NIH 3T3 cells, it potently activated isoform-specific p38alpha MAP kinase signaling and weakly stimulated ERK signaling in pheochromocytoma (PC6) cells. This cell-type specificity is explained in part by the finding that Rin binds and stimulates b-Raf but does not activate c-Raf. Accordingly, selective down-regulation of Rin in PC6 cells suppressed neurotrophin-elicited activation of b-Raf and p38, without obvious effects on NGF-induced ERK activation. Moreover, the ability of NGF to promote neurite outgrowth was inhibited by Rin knockdown. Together, these observations establish Rin as a neuronal specific regulator of neurotrophin signaling, required to couple NGF stimulation to sustain activation of p38 MAP kinase and b-Raf signaling cascades required for neuronal development.