R-Ras regulation of integrin function.
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Biomedical subjects
B Oertli
Publications and source records attributed to B Oertli.
Hyaluronan-induced cyclooxygenase-2 expression promotes thromboxane A2 production by renal cells.
BACKGROUND: Matrix degradation products such as fragmented hyaluronan (HA) display important proinflammatory effects on renal tubular epithelial cells (TECs) and macrophages (MPhis). We hypothesized that HA could up-regulate cyclooxygenase type 2 (COX-2) in these cells and that the subsequent production of thromboxane A2 (TXA2) could play a role in inflammatory renal lesions. METHODS: We used an in vitro approach to examine the expression of COX-1 and COX-2 and the production of TXA2 in response to fragments of HA. COX-2 mRNA, protein, and the resulting TXA2 production were measured in CD44-positive, HA-responsive cells lines of TECs and MPhi. COX-2 mRNA was also measured in vivo in MRL-Faslpr mice and in mice with anti-glomerular basement membrane (anti-GBM) nephritis. RESULTS: In TECs and MPhis, HA increased the steady-state COX-2 mRNA and protein levels markedly, whereas COX-1 mRNA levels did not change. The HA-induced response was comparable to lipopolysaccharide stimulation. In comparison with MPhi, the response was much weaker in TECs. Likewise, the production of TXA2 in response to HA was markedly increased in MPhi, but less in TECs. In TECs and in MPhi, the HA-stimulated TXA2 synthesis was inhibited with the COX-2-selective inhibitors SC58125 (12.5 micromol/L) or celecoxib (0.25 to 5.00 micromol/L). COX-2 mRNA levels were increased in nephritic mice with MRL-Faslpr lupus nephritis and in mice with anti-GBM disease. CONCLUSIONS: HA is a proinflammatory factor that stimulates COX-2 expression and subsequent TXA2 production. Since HA accumulates markedly in renal injury, we speculate that this matrix molecule could therefore play a significant role in thromboxane-mediated immune events in the kidney.
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The effector loop and prenylation site of R-Ras are involved in the regulation of integrin function.
The closely related small GTP-binding proteins H-Ras and R-Ras have opposing effects on the regulation of integrin cell adhesion receptors. To gain insight into the properties of R-Ras with respect to the regulation of integrin function and interactions with downstream effectors we performed an analysis of R-Ras variants containing mutations in the effector binding domain and C-terminal prenylation site. We found that the activation of the downstream effector PI 3-kinase was sensitive to mutations in the effector binding domain, as was the binding to the effectors, Ral-GDS, Raf-1 and the novel effector Nore1. Furthermore, specific mutations in the effector binding loop and C-terminal prenylation motif impaired the ability of R-Ras to regulate integrin function in CHO cells. However, the ability of the R-Ras effector loop mutants to bind, and activate known effectors did not correlate with their ability to regulate integrin function. Thus, the known R-Ras effectors are not critical for regulating integrin activation, at least in CHO cells. Consequently, these studies provide insight into the structural basis of the interactions between R-Ras and its candidate effectors and suggest the existence of novel mechanisms through which this GTPase could regulate cell adhesion.
Amino Acid Sequence↗
Lipopolysaccharide induces functional ICAM-1 expression in rat alveolar epithelial cells in vitro.
Lipopolysaccharide (LPS)-induced lung inflammation is known to increase pulmonary intercellular adhesion molecule-1 (ICAM-1) expression. In the present study, L2 cells, a cell line of alveolar epithelial cells, were stimulated with LPS, and ICAM-1 expression was studied. ICAM-1 protein on L2 cells peaked at 6 (38% increase; P < 0.01) and 10 (48% increase; P < 0.001) h after stimulation with Escherichia coli and Pseudomonas aeruginosa LPS, respectively. ICAM-1 mRNA expression was markedly increased, with a peak at 2-4 (E. coli) and 4-6 (P. aeruginosa) h. Adherence assays of neutrophils to LPS-stimulated L2 cells showed a threefold increase in adherence (P < 0.001). Pretreatment of the neutrophils with anti-lymphocyte function-associated antigen-1 and anti-Mac-1 antibodies reduced adherence by 54% (P < 0.001). Analysis of immunofluorescence staining for ICAM-1 showed an exclusive apical expression of ICAM-1. These results indicate that LPS upregulates functional active ICAM-1 on the apical part of the membrane in rat pneumocytes.
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Regulation of hyaluronan-stimulated VCAM-1 expression in murine renal tubular epithelial cells.
BACKGROUND: Cytokines stimulate the expression of the adhesion molecule VCAM-1 in renal tubular epithelial cells. We have recently shown that VCAM-1 can also be upregulated by low molecular weight breakdown products of the matrix constituent hyaluronan (HA) (J Immunol 1998; 161: 3431-3437). The mechanisms of VCAM-I expression in response to HA remain to be defined. METHODS: Using a defined mouse cortical tubular (MCT) cell line we investigated the effect of protein kinase C (PKC) and tyrosine kinase (TK) inhibition on the HA-stimulated VCAM-1 expression by cell ELISA and RT PCR or Northern blotting. Furthermore, we examined the effect of PKC and TK inhibition on NF-kappaB. RESULTS: We found that the PKC inhibitor GF109203X (acting on conventional, novel and atypical isoforms) inhibited the HA-stimulated VCAM-1 expression in MCT cells dose-dependently up to 90%, whereas chelerythrine (acting on conventional and novel isoforms) had no effect. Downregulation of PKC with PMA did not prevent the HA-stimulated VCAM-1 expression, suggesting that Ca2+- and diacylglycerol-independent (atypical) isoforms of PKC are involved. The TK inhibitor genistein also inhibited the HA-stimulated VCAM-1 expression at the mRNA and protein level up to 70%. Interestingly, the HA-stimulated nuclear translocation of NF-kappaB could not be prevented with GF109203X and genistein. CONCLUSION: These data demonstrate that the HA-stimulated VCAM-1 expression in MCT cells involves PKC and TK pathways. The absence of an effect of PKC and TK inhibitors on the nuclear translocation of NF-kappaB suggests that additional transcription factors are involved for VCAM-1 expression.
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Enhanced hyaluronan synthesis in the MRL-Fas(lpr) kidney: role of cytokines.
BACKGROUND/AIM: Pathological accumulation of the extracellular matrix component hyaluronan (HA) occurs in the kidney cortex in immune-system mediated tissue injury. The purpose of the present study was to examine the pattern of HA deposition and the mechanisms of HA synthesis in the MRL-Fas(lpr) mouse model of lupus nephritis. METHODS: Kidneys from normal and autoimmune mice were examined for HA content by immunofluorescence staining. Steady state mRNA levels for key enzymes involved in HA synthesis - uridine diphosphate-glucose dehydrogenase (UDPGDH) and HA synthases (HAS) 1, 2 and 3 - were assessed by reverse-transcriptase polymerase chain reaction (RT-PCR). Using cultured mouse tubular epithelial cells, the regulation of the HA production in vitro in response to tumor necrosis factor alpha and interferon gamma was also examined. RESULTS: By immunofluorescence staining, large amounts of HA were detected in the cortical interstitium of MRL-Fas(lpr) mice with autoimmune renal injury, but not in congenic MRL-++ mice. By RT-PCR the presence of transcripts for several genes involved in the synthesis of HA in normal and autoimmune kidneys could be demonstrated, including mRNA for UDPGDH and HAS1 and HAS2, but not for HAS3. Except for HAS2, steady state mRNA levels for these enzymes did not correlate with disease activity. Analyzing a kidney tubular epithelial cell line in vitro, it was found that tumor necrosis factor alpha and interferon gamma, and particularly the combination of these two cytokines, markedly enhanced the synthesis of HA. The expression of HAS2 mRNA was also enhanced in response to cytokine treatment. CONCLUSIONS: HA deposition is prominent in MRL-Fas(lpr) mice with renal disease and could be mediated by local synthesis through HAS1 and HAS2. We hypothesize that the enhanced synthesis of HA could be promoted by proinflammatory cytokines in vivo. The functional significance of HA accumulation in autoimmune renal injury remains to be determined.
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Mechanisms of hyaluronan-induced up-regulation of ICAM-1 and VCAM-1 expression by murine kidney tubular epithelial cells: hyaluronan triggers cell adhesion molecule expression through a mechanism involving activation of nuclear factor-kappa B and activating protein-1.
The matrix constituent hyaluronan (HA) markedly accumulates in inflammatory lesions. To gain insight into the biologic significance of this phenomenon we tested the hypothesis that HA could regulate cell adhesion molecule expression in epithelial cells. Using a clonal line of mouse cortical tubular (MCT) cells we found that fragmented intermediate m.w., but not high m.w., HA markedly increased ICAM-1 and VCAM-1 steady state mRNA and cell surface expression. Up-regulation of ICAM-1 and VCAM-1 mRNA by HA was preceded by a marked increase in NF-kappaB and activating protein-1 DNA binding activity in MCT cells. Transcript levels for the NF-kappaB inhibitor IkappaBalpha and for the activating protein-1 constituents c-jun and c-fos also increased in response to HA stimulation of tubular cells. Inhibition of NF-kappaB with the serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone blocked the HA-mediated expression of ICAM-1 and VCAM-1 in MCT cells. In conclusion, HA displays proinflammatory effects by directly stimulating the expression of the cell adhesion molecules ICAM-1 and VCAM-1 in mouse kidney epithelial cells. HA could thereby play an important role in leukocyte adhesion in inflammatory renal diseases.
3T3 Cells↗
Characterization of CD44-mediated hyaluronan binding by renal tubular epithelial cells.
BACKGROUND: CD44 is the main receptor for the extracellular polysaccharide hyaluronan (HA). We have recently shown that CD44 is strongly induced on renal tubular epithelial cells (TEC) in autoimmune renal injury and that HA accumulates in the renal interstitium (Kidney Int 1996; 50: 156-163 and Nephrol Dial Transplant 1997; 12: 1344-1353). The functional significance of enhanced tubular CD44 expression and its interaction with HA are not known. The purpose of the present study was to characterize renal tubular CD44 expression and CD44-mediated HA binding in vitro and to investigate the growth modulating effects in response to HA binding by TEC. METHODS: RT-PCR analysis, flow cytometry, confocal microscopy and Western blotting were used to examine cell surface and soluble CD44 expression by cultured TEC, using SV40-transformed mouse cortical tubular (MCT) cells. HA binding characteristics were examined by flow cytometry and effects of HA on TEC cell growth by [3H]thymidine incorporation. RESULTS: By RT-PCR analysis MCT cells expressed predominantly the standard form of CD44 mRNA, whereas the expression of variant forms was very weak. Confocal microscopy showed that CD44 was expressed basolaterally and apically on MCT cells with strong staining on microvilli. Shedding of CD44 from MCT cells could be induced with crosslinking of anti-CD44 mAbs or with PMA stimulation. MCT cells constitutively bound HA and this binding could be modulated with anti-CD44 mAbs. Soluble and plate-bound HA markedly inhibited MCT cell growth. CONCLUSIONS: CD44 is a regulated HA receptor on MCT cells which can be shed into the cellular environment. Upon binding of HA, CD44 functions as a growth inhibitory cell surface protein in MCT cells. We speculate that the interaction of CD44 with HA may have important regulatory effects on cell proliferation in tubulointerstitial renal diseases.
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Characteristic matrix and tubular basement membrane abnormalities in the CBA/Ca-kdkd mouse model of hereditary tubulointerstitial disease.
CBA/CaH-kdkd mice develop hereditary tubulointerstitial disease with mononuclear cell infiltration and cyst formation, possibly representing a model of human nephronophthisis. The purpose of the present investigation was to examine the components of the fibrotic changes which typically develop in the kidneys of these mice. By conventional histology, kdkd mice displayed progressive interstitial fibroblast and matrix accumulation. Immunohistological analysis of kdkd kidneys showed marked deposition of fibronectin in the tubulointerstitial space and revealed prominent irregularities for laminin and collagen type IV in the tubular basement membrane (TBM), including thickening, widening and folding. Electron microscopy confirmed the TBM abnormalities and showed marked undulation and thickening in areas of proximal tubular (PT) degeneration. Immunofluorescence staining analysis for the fibronectin receptors VLA-4 and VLA-5 showed no expression on injured proximal tubules, whereas the expression of the laminin receptor VLA-6 was increased and irregular on altered PT. Analysis of RNA derived from kdkd kidneys revealed marked upregulation of steady-state mRNA levels for the fibrogenic growth factor TGF-beta. We conclude that TBM alterations, matrix accumulation and changes in integrin expression together with enhanced TGF-beta production are typical features of kdkd tubulointerstitial disease and suggest that characteristic TBM or matrix alterations could contribute to the pathogenesis of the disease in these mice.
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Hyaluronan induces monocyte chemoattractant protein-1 expression in renal tubular epithelial cells.
Hyaluronan (HA) is a nonsulfated glycosaminoglycan that accumulates in the renal interstitium in immune-mediated kidney diseases. The functional significance of such HA deposition in the kidney has not been elucidated. Several studies have suggested that HA may exhibit proinflammatory effects. Since chemokines such as monocyte chemoattractant protein-1 (MCP-1) play an important role in the recruitment of leukocytes in renal injury, this study tested whether HA and its fragments could promote MCP-1 production by renal parenchymal cells. Mouse cortical tubular cells were stimulated with fragmented HA or with high molecular weight HA (Healon) in vitro and were examined for MCP-1 expression. Fragmented HA, but not Healon, increased MCP-1 mRNA within 30 min with a peak after 2 h. In addition, a 10-fold increase of MCP-1 protein in the supernatant was found after a 6-h stimulation with fragmented HA. The enhanced MCP-1 mRNA and protein expression in response to HA was dose-dependent between 1 and 100 microg/ml. Upregulation of MCP-1 protein production could be blocked by preincubation with actinomycin D or cycloheximide, suggesting that MCP-1 mRNA and protein expression in response to HA are based on de novo synthesis. The HA-stimulated MCP-1 production was also inhibited with anti-CD44 antibodies, suggesting that MCP-1 is upregulated at least in part by signaling through CD44. In summary, fragmented HA markedly stimulates renal tubular MCP-1 production by mechanisms that involve binding to the HA receptor CD44. It is hypothesized that the accumulation of HA in immune renal injury could participate in the recruitment and activation of inflammatory cells in vivo through production of MCP-1.
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Up-regulation of tubular epithelial interleukin-12 in autoimmune MRL-Fas(lpr) mice with renal injury.
Phagocyte-derived interleukin-12 (IL-12) is a key cytokine that induces the development of an effective Th1 type immune response in various inflammatory and infectious disorders. To determine the importance of IL-12 in the pathogenesis of autoimmune renal injury we examined the renal production of this heterodimeric cytokine in the MRL-Fas(lpr) lupus nephritis model. Compared with normal mice RT-PCR products encoding both the p35 and p40 subunits of IL-12 were markedly increased in the kidney of MRL-Fas(lpr). Immunofluorescence staining demonstrated expression of the IL-12 p75 heterodimer on isolated infiltrating mononuclear cells and also on proximal tubular epithelial cells in MRL-Fas(lpr) but less in normal mice kidneys. The enhanced expression of IL-12 correlated with an increased intrarenal transcription of IFN-gamma. The p35 and p40 transcripts and soluble IL-12 p75 protein were also produced by cultured TEC. In addition, membrane bound IL-12 was detected on Tec. We conclude that IL-12 production is significantly up-regulated in MRL-Fas(lpr) lupus nephritis. In addition to mononuclear cells, TEC are an important source of IL-12 and could thereby participate in the development of a Th1 type immune response in autoimmune renal injury.
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Identification of a renal proximal tubular epithelial antigen coexpressed on leukocytes.
Renal tubular epithelial cells (TEC) and leukocytes express distinct cell adhesion molecules and integrins in common. To identify additional proximal TEC molecules that are coexpressed on leukocytes, we have generated a panel of monoclonal antibodies (mAb) by immunization of rats with a mouse T cell line. One of these mAb (I-72) recognizes a 210-kD antigen on cultured TEC. The staining pattern in several cell lines allowed exclusion of known TEC surface molecules as being the antigens recognized by the I-72 mAb. Immunofluorescence staining of mouse kidney with the I-72 mAb demonstrated prominent expression on the apical but not the basolateral side of proximal tubules, predominantly in the S1 segment. I-72 was also expressed in the loop of Henle, the collecting duct, and on endothelial cells of intrarenal vessels. We have thus generated a mAb which identifies an unusual renal proximal tubular brush border molecule that is coexpressed on T cells. Molecular structure and the function of I-72 need to be defined in further studies.
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