Bradykinin decreases podocyte permeability through ADAM17-dependent epidermal growth factor receptor activation and zonula occludens-1 rearrangement.

Recent data show that increases in bradykinin (BK) concentration contribute to the beneficial effects of angiotensin-converting enzyme inhibitor (ACEI) treatment in chronic kidney disease. However, the possible role of BK in attenuated proteinuria, often seen in ACEI-treated patients, is not well studied. Here, we report that BK decreases mouse podocyte permeability through rearrangement of the tight junction protein zonula occludens-1 (ZO-1) and identify some of the major signaling events leading to permeability change. We show that BK2 receptor (BK2R) stimulation transactivates the epidermal growth factor receptor (EGFR). EGFR transactivation is mediated by a disintegrin and metalloenzyme (ADAM) family members, which are required for both extracellular signal-regulated kinase (ERK) and EGFR activation by BK. Using a gene-silencing approach we observed that both BK-induced ERK activation and BK-induced permeability decrease in podocytes is attenuated by ADAM17 down-regulation, and we identified epiregulin (ER) as the EGFR ligand participating in ADAM-dependent BK2R-EGFR cross-talk. EGFR inhibition attenuated both ZO-1 rearrangement and BK-induced permeability decreases in podocyte. We propose that ZO-1 redistribution is an important element of BK-induced permeability change and the signaling events involved in ZO-1 rearrangement include transactivation of the EGFR via ADAM17 activation and ER shedding. Our data indicate that ADAM17 and the EGFR may be potential novel therapeutic targets in diabetic nephropathy and other chronic kidney diseases.

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.

ADAM-17 regulates endothelial cell morphology, proliferation, and in vitro angiogenesis.

Modulation of angiogenesis is a promising approach for treating a wide variety of human diseases including ischemic heart disease and cancer. In this study, we show that ADAM-17 is an important regulator of several key steps during angiogenesis. Knocking down ADAM-17 expression using lentivirus-delivered siRNA in HUVECs inhibited cell proliferation and the ability of cells to form close contact in two-dimensional cultures. Similarly, ADAM-17 depletion inhibited the ability of HUVECs to form capillary-like networks on top of three-dimensional Matrigel as well as in co-culture with fibroblasts within a three-dimensional scaffold. In mechanistic studies, both baseline and VEGF-induced MMP-2 activation and Matrigel invasion were inhibited by ADAM-17 depletion. Based on our findings we propose that ADAM-17 is part of a novel pro-angiogenic pathway leading to MMP-2 activation and vessel formation.

Endogenous ouabain-like factor (OLF) secretion is modulated by nicotinic mechanisms in rat adrenocortical cells.

This study tested the hypothesis that rat adrenocortical secretion of endogenous ouabain-like factor (OLF) is regulated by nicotinic mechanisms. OLF secreted by dispersed cell suspensions of zona glomerulosa (ZG) and fasciculata/reticularis (ZFR) cells was found to co-elute with authentic ouabain by reverse phase HPLC; OLF concentrations in cell supernatants were measured by radioimmunoassay. Nicotine (10(-6) - 10(-3) M) stimulated significant OLF secretion in rat adrenocortical cells. Acetylcholine (10(-7) - 10(-4) M) and eserine (10(-7) - 10(-3) M) stimulated OLF secretion in ZG cells at lower concentrations and stimulated at higher concentrations. Acetylcholine had no effect on ZFR secretion of OLF, but eserine stimulated OLF secretion. ACTH (10(-8) M) strongly potentiated the OLF stimulatory effect of nicotine in ZG cells; however significant interactions between nicotine and ACTH or angiotensin II on OLF secretion in ZFR cells were not apparent. The ganglionic blockers hexamethonium and mecamylamine further potentiated the effect of nicotine, implicating nicotinic acetylcholine receptors (nAChRs) in regulation of OLF secretion. The alpha7-receptor antagonist methyllycaconitine (MLA) dose-dependently inhibited the effect of nicotine in the ZG cells, and in ZFR cells MLA potentiated nicotine-induced OLF secretion. These data suggest that nicotinic regulation may underlie OLF secretion by rat adrenocortical cells, and strongly suggest presence of functional nicotinic acetylcholine receptors on these cells.

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 α5ß1 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 α5ß1 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 α5ß1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5ß1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5ß1 integrin interaction. In cell-free assays, we showed that purified α5ß1 integrin and ß1 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 ß1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of ß1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing ß1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT. CONCLUSIONS/SIGNIFICANCE: Our data show for the first time that ß1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5ß1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.

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.

5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-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-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) 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-HT(2A) 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.

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.

Curcumin-induced apoptosis in scleroderma lung fibroblasts: role of protein kinase cepsilon.

Scleroderma, a disease involving excessive collagen deposition, can be studied using fibroblasts cultured from affected tissues. We find that curcumin, the active component of the spice turmeric, causes apoptosis in scleroderma lung fibroblasts (SLF), but not in normal lung fibroblasts (NLF). This effect is likely to be linked to the fact that although curcumin induces the expression of the phase 2 detoxification enzymes heme oxygenase 1 and glutathione S-transferase P1 (GST P1) in NLF, SLF are deficient in these enzymes, particularly after curcumin treatment. The sensitivity of cells to curcumin-induced apoptosis and the expression of GST P1 (but not heme oxygenase 1) are regulated by the epsilon isoform of protein kinase C (PKCepsilon). SLF, which contain less PKCepsilon and less GST P1 than NLF, become less sensitive to curcumin-induced apoptosis and express higher levels of GST P1 when transfected with wild-type PKCepsilon, but not with dominant-negative PKCepsilon. Conversely, NLF become sensitive to curcumin-induced apoptosis and express lower levels of GST P1 when PKCepsilon expression or function is inhibited. The subcellular distribution of PKCepsilon also differs in NLF and SLF. PKCepsilon is predominantly nuclear or perinuclear in NLF but is associated with stress fibers in SLF. Just as PKCepsilon levels are lower in SLF than in NLF in vitro, PKCepsilon expression is decreased in fibrotic lung tissue in vivo. In summary, our results suggest that a signaling pathway involving PKCepsilon and phase 2 detoxification enzymes provides protection against curcumin-induced apoptosis in NLF and is defective in SLF. These observations suggest that curcumin may have therapeutic value in treating scleroderma, just as it has already been shown to protect rats from lung fibrosis induced by a variety of agents.