Pal Gooz

Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo.

Lung fibrosis involves the overexpression of ECM proteins, primarily collagen, by alpha-smooth muscle actin (ASMA)-positive cells. Caveolin-1 is a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the caveolin-1 scaffolding domain (CSD peptide) inhibits collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from the fibrotic lungs of scleroderma patients (SLF). CSD peptide inhibits ASMA expression in SLF but not NLF. Similar inhibition of collagen, tenascin-C, and ASMA expression was also observed when caveolin-1 expression was upregulated using adenovirus. These observations suggest that the low caveolin-1 levels in SLF cause their overexpression of collagen, tenascin-C, and ASMA. In mechanistic studies, MEK, ERK, JNK, and Akt were hyperactivated in SLF, and CSD peptide inhibited their activation and altered their subcellular localization. These studies and experiments using kinase inhibitors suggest many differences between NLF and SLF in signaling cascades. To validate these data, we determined that the alterations in signaling molecule activation observed in SLF also occur in fibrotic lung tissue from scleroderma patients and in mice with bleomycin-induced lung fibrosis. Finally, we demonstrated that systemic administration of CSD peptide to bleomycin-treated mice blocks epithelial cell apoptosis, inflammatory cell infiltration, and changes in tissue morphology as well as signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. CSD peptide may be a prototype for novel treatments for human lung fibrosis that act, in part, by inhibiting the expression of ASMA and ECM proteins.

Opposing Effects of Protein Kinase Cα and Protein Kinase Cϵ on Collagen Expression by Human Lung Fibroblasts Are Mediated via MEK/ERK and Caveolin-1 Signaling

The roles of MEK, ERK, the ϵ and α 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 PKCϵ leads to MEK/ERK activation. In another, increased PKCα 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 PKCϵ, PKCα, 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, PKCϵ, PKCα, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.

5-HT2A Receptor Induces ERK Phosphorylation and Proliferation through ADAM-17 Tumor Necrosis Factor-α-converting Enzyme (TACE) Activation and Heparin-bound Epidermal Growth Factor-like Growth Factor (HB-EGF) Shedding in Mesangial Cells

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-α-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT2A receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT → 5-HT2A receptor → TACE → HB-EGF shedding → EGFR → ERK → increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.

Interleukin 1β induces gastric epithelial cell matrix metalloproteinase secretion and activation during Helicobacter pylori infection

Background and aims: Matrix metalloproteinases (MMPs) are endopeptidases with roles in extracellular matrix remodelling, cell proliferation, and inflammatory processes. We showed previously that Helicobacter pylori infection of human gastric adenocarcinoma (AGS) cells increased epithelial secretion of epithelial MMP-1 and MMP-3 and bacterial secretion of MMP-3-like activity. In the present study, we sought to characterise the role of interleukin (IL)-1β in H pylori induced secretion of epithelial MMPs. Methods and results: AGS cells were treated with H pylori and/or IL-1β. Comparable IL-8 secretory responses (∼1700 ng/ml) measured by ELISA were induced by 2.0 ng/ml IL-1β and by H pylori at a multiplicity of infection (MOI) of 50. The same IL-1β and H pylori concentrations induced comparable increases in AGS cell caseinolytic activity at 60 kDa. MMP-3 monoclonal antibody immunoblots of AGS cell conditioned media detected immunoreactive bands at 71 kDa and 56 kDa. H pylori (MOI=50–100) induced dose dependent increases in both bands whereas IL-1β (0.2–2 ng/ml) induced dose dependent increases only in the 71 kDa band, which was identified as a MMP-3/TIMP-3 (tissue inhibitor of metalloproteinases 3) heterodimer. AGS/H pylori conditioned media expressed 24 times more MMP-3 activity than AGS/IL-1β conditioned media. There was a strong interaction between IL-1β and H pylori on MMP-3 secretion. Conclusions: We conclude that IL-1β induces gastric epithelial cell MMP-3 secretion, contributing to epithelial tissue destruction during H pylori infection. However, other bacterial/host factors are needed to mediate the full gastric epithelial cell MMP-3 secretory response induced by H pylori infection.

Caveolin-1 regulates leucocyte behaviour in fibrotic lung disease

Objectives Reduced caveolin-1 levels in lung fibroblasts from patients with scleroderma and the lungs of bleomycin-treated mice promote collagen overexpression and lung fibrosis. This study was undertaken to determine whether caveolin-1 is deficient in leucocytes from bleomycin-treated mice and patients with scleroderma and to examine the consequences of this deficiency and its reversal. Methods Mice or cells received the caveolin-1 scaffolding domain (CSD) peptide to reverse the pathological effects of reduced caveolin-1 expression. In bleomycin-treated mice, the levels of caveolin-1 in leucocytes and the effect of CSD peptide on leucocyte accumulation in lung tissue were examined. To validate the results in human disease and to identify caveolin-1-regulated molecular mechanisms, monocytes and neutrophils were isolated from patients with scleroderma and control subjects and caveolin-1, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38, CXC chemokine receptor 4 (CXCR4) and matrix metalloproteinase 9 (MMP-9) expression/activation were evaluated. These parameters were also studied in monocytes treated with cytokines or CSD peptide. Results Leucocyte caveolin-1 is important in lung fibrosis. In bleomycin-treated mice, caveolin-1 expression was diminished in monocytes and CSD peptide inhibited leucocyte recruitment into the lungs. These observations are relevant to human disease. Monocytes and neutrophils from patients with scleroderma contained less caveolin-1 and more activated ERK, JNK and p38 than those from control subjects. Treatment with CSD peptide reversed ERK, JNK and p38 hyperactivation. Scleroderma monocytes also overexpressed CXCR4 and MMP-9, which was inhibited by the CSD peptide. Cytokine treatment of normal monocytes caused adoption of the scleroderma phenotype (low caveolin-1, high CXCR4 and MMP-9 and signalling molecule hyperactivation). Conclusions Caveolin-1 downregulation in leucocytes contributes to fibrotic lung disease, highlighting caveolin-1 as a promising therapeutic target in scleroderma.

TRPP2 and TRPV4 Form an EGF-Activated Calcium Permeable Channel at the Apical Membrane of Renal Collecting Duct Cells

Objective Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear. Methods and Results We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium. Conclusion We conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

A Disintegrin and Metalloenzyme (ADAM) 17 Activation Is Regulated by α5β1 Integrin in Kidney Mesangial Cells

Background The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation. Methodology/Principal Findings We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT. Conclusions/Significance Our data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.

Role of Cytokines in Helicobacter pylori-Induced Gastric Epithelial Cell Matrix Metalloproteinase Secretion and Activation

Initial responses to gastric colonization by Helicobacter pylori include a transient period of achlorhydria and activation of neutrophils (1). In our previous work (2) we showed that acute bacterial infection of human gastric adenocarcinoma cells (AGS) inhibits the activity of human H,K-ATPase α subunit promoter, which may contribute to lowered acid output found in acute H. pylori infection in vivo. The inflammatory response is hypothesized to be induced both directly by bacterial factors like LPS, urease, porins, etc and indirectly by induction of the chemokine IL-8 and cytokines (IL-1, TNFα), leading to mucosal damage and ulceration (1). Matrix metalloproteinases play major roles in tissue degradation, cell migration and also inflammatory processes. We have previously shown that H. pylori infection of AGS cells increased secretion of epithelial MMPs-1 and -3 and bacterial MMP-3-like activity (3). Our aim in the present study was to characterize the relationship between activation of matrix metalloproteinases and IL-1β and TNFα, potent stimulators of IL-8 secretion in gastric epithelia during H pylori infection.