Christopher A. McCulloch

The compliance of collagen gels regulates transforming growth factor-β induction of α-smooth muscle actin in fibroblasts

Wound contraction is mediated by myofibroblasts, specialized fibroblasts that appear in large numbers as the wound matures and when resistance to contractile forces increases. We considered that the regulation of myofibroblast differentiation by wound-healing cytokines may be dependent on the resistance of the connective tissue matrix to deformation. We examined transforming growth factor-β1 (TGF-β1) induction of the putative fibroblast contractile marker, α-smooth muscle actin (α-SMA), and the regulation of this process by the compliance of collagen substrates. Cells were cultured in three different types of collagen gels with wide variations of mechanical compliance as assessed by deformation testing. The resistance to collagen gel deformation determined the levels of intracellular tension as shown by staining for actin stress fibers. For cells plated on thin films of collagen-coated plastic (ie, minimal compliance and maximal intracellular tension), TGF-β1 (10 ng/ml; 6 days) increased α-SMA protein content by ninefold as detected by Western blots but did not affect β-actin content. Western blots of cells in anchored collagen gels (moderate compliance and tension) also showed a TGF-β1-induced increase of α-SMA content, but the effect was greatly reduced compared with collagen-coated plastic (<3-fold increase). In floating collagen gels (high compliance and low tension), there were only minimal differences of α-SMA protein. Northern analyses for α-SMA and β-actin indicated that TGF-β1 selectively increased mRNA for α-SMA similar to the reported protein levels. In pulse-chase experiments, [35S]methionine-labeled intracellular α-SMA decayed most rapidly in floating gels, less rapidly in anchored gels, and not at all in collagen plates after TGF-β1 treatment. TGF-β1 increased α2 and β1 integrin content by 50% in cells on collagen plates, but the increase was less marked on anchored gels and was undetectable in floating gels. When intracellular tension on collagen substrates was reduced by preincubating cells with blocking antibodies to the α2 and β1 integrin subunits, TGF-β1 failed to increase α-SMA protein content in all three types of collagen matrices. These data indicate that TGF-β1-induced increases of α-SMA content are dependent on the resistance of the substrate to deformation and that the generation of intracellular tension is a central determinant of contractile cytoskeletal gene expression.

Reproducibility and Sensitivity of Oral Malodor Measurements with a Portable Sulphide Monitor

Forty-one subjects with bad breath were assessed for oral malodor and periodontal status on three occasions, at intervals of approximately one week. Oral malodor was assessed by measurement of peak and steady-state volatile sulphide levels with a portable sulphide monitor and by organoleptic measurement of whole-mouth, tongue dorsum, and interproximal dental odors by two independent judges. Reproducibility of measurements, assessed by paired t tests and Kappa testing, demonstrated no significant differences between any ofthe test results from the first and second appointments. Steady-state sulphide levels were the most reproducible of all tests. The ability of the tests to detect an expected reduction of malodor following a 0.2% chlorhexidine mouthrinse regimen was investigated by comparison of test values between the second and third appointments. Following the mouthrinsing treatment, 43% reductions of peak, 47% reductions of steady-state volatile sulphide levels, and 15-58% reductions in all other measurement categories were observed. The majority of the participants (22/41) had no pockets greater than 5 mm and exhibited both moderate gingival inflammation (Mean Gingival Index = 1.17) and moderate plaque accumulation (Mean Plaque Index = 1.84). Plaque Index and presence of pockets greater than 7 mm were weakly related to sulphide measurements. Whereas assessment of steady-state sulphide levels by the sulphide monitor does not constitute a direct measure of oral malodor, its relation to organoleptic measurement, superior reproducibility, objectivity, and sensitivity support the use of the sulphide monitor in clinical studies.

Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration.

Osteopontin (OPN) is a secreted glycoprotein with mineral‐ and cell‐binding properties that can regulate cell activities through integrin receptors. Previously, we identified an intracellular form of osteopontin with a perimembranous distribution in migrating fetal fibroblasts (Zohar et al., J Cell Physiol 170:88–98, 1997). Since OPN and CD44 expression are increased in migrating cells, we analyzed the relationship of these proteins with immunofluorescence and confocal microscopy. A distinct co‐localization of perimembranous OPN and cell‐surface CD44 was observed in fetal fibroblasts, periodontal ligament cells, activated macrophages, and metastatic breast cancer cells. The co‐localization of OPN and CD44 was prominent at the leading edge of migrating fibroblasts, where OPN also co‐localized with the ezrin/radixin/moesin (ERM) protein ezrin, as well as in cell processes and at attachment sites of hyaluronan‐coated beads. The subcortical location of OPN in these cells was verified by cell‐surface biotinylation experiments in which biotinylated CD44 and non‐biotinylated OPN were isolated from complexes formed with hyaluronan‐coated beads and identified with immunoblotting. That perimembranous OPN represents secreted protein internalized by endocytosis or phagocytosis appeared to be unlikely since exogenous OPN that was added to cell cultures could not be detected inside the cells. A physical association with OPN, CD44, and ERM, but not with vinculin or α‐actin, was indicated by immunoadsorption and immunoblotting of cell proteins in complexes extracted from hyaluronan‐coated beads. The functional significance of OPN in this complex was demonstrated using OPN−/− and CD−/− mouse fibroblasts which displayed impaired migration and a reduced attachment to hyaluronan‐coated beads. These studies indicate that OPN exists as an integral component of a hyaluronan‐CD44‐ERM attachment complex that is involved in the migration of embryonic fibroblasts, activated macrophages, and metastatic cells. J. Cell. Physiol. 184:118–130, 2000.

Periodontal ligament cell populations: The central role of fibroblasts in creating a unique tissue

Fibroblasts are the predominant cells of the periodontal ligament (PL) and have important roles in the development, function, and regeneration of the tooth support apparatus. Biological processes initiated during the formation of the PL contribute to the long‐lasting homeostasic properties exhibited by PL fibroblast populations.

Force activates smooth muscle α-actin promoter activity through the Rho signaling pathway

In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle α-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho–Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.65 pN/μm2) to integrins via collagen-coated magnetite beads. Force maximally induced RhoA activation at 10 minutes that was localized to force application sites and required intact actin filaments. Force application induced phosphorylation of LIM kinase (5-10 minutes) and an early dephosphorylation of cofilin (5 minutes) that was followed by prolonged cofilin phosphorylation. These responses were blocked by Y27632, an inhibitor of Rho kinase. Force promoted actin filament assembly at force application sites (10-20 minutes), a process that required Rho kinase and cofilin. Force application induced nuclear translocation of the transcriptional co-activator MRTF-A but not MRTF-B. Nuclear translocation of MRTF-A required Rho kinase and intact actin filaments. Force caused 3.5-fold increases of SMA promoter activity that were completely blocked by transfection of cells with dominant-negative MRTF-A or by inhibition of Rho kinase or by actin filament disassembly. These data indicate that mechanical forces mediate actin assembly through the Rho–Rho-kinase–LIMK cofilin pathway. Force-mediated actin filament assembly promotes nuclear translocation of MRTF and subsequent activation of the SMA promoter to enhance SMA expression.

The autoimmune disease-associated PTPN22 variant promotes calpain-mediated Lyp/Pep degradation associated with lymphocyte and dendritic cell hyperresponsiveness

A variant of the PTPN22-encoded Lyp phosphatase (Lyp620W) confers risk for autoimmune disease, but the mechanisms underlying this association remain unclear. We show here that mice expressing the Lyp variant homolog Pep619W manifest thymic and splenic enlargement accompanied by increases in T-cell number, activation and positive selection and in dendritic- and B-cell activation. Although Ptpn22 (Pep) transcript levels were comparable in Pep619W and wild-type Pep619R mice, Pep protein levels were dramatically reduced in the mutant mice, with Pep619W protein being more rapidly degraded and showing greater association with and in vitro cleavage by calpain 1 than Pep619R. Similarly, levels of the Lyp620W variant were decreased in human T and B cells, and its calpain binding and cleavage were increased relative to wild-type Lyp620R. Thus, calpain-mediated degradation with consequently reduced Lyp/Pep expression and lymphocyte and dendritic cell hyperresponsiveness represents a mechanism whereby Lyp620W may increase risk for autoimmune disease.

Integrity of cell-cell contacts is a critical regulator of TGF-β1-induced epithelial-to-myofibroblast transition: Role for β-catenin

Injury of the tubular epithelium and TGF-β1-induced conversion of epithelial cells to α-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-β1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-β1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca2+-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-β1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since β-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-β1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and β-catenin, but TGF-β1 selectively rescued β-catenin and stimulated the β-catenin-driven reporter TopFLASH. Moreover, chelation of free β-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-β1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a β-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-β1 are required for EMT.

Osteopontin Expression Is Required for Myofibroblast Differentiation

Osteopontin (OPN) is a multifunctional cytokine that is strongly expressed in healing wounds and fibrotic lesions, both of which are characterized by the formation of myofibroblasts. We examined the role of OPN in myofibroblast differentiation induced by the profibrotic cytokine transforming growth factor-β1. In cultured cardiac or dermal fibroblasts treated with transforming growth factor-β1, there was a 2- to 5-fold increase in the expression of the myofibroblast markers α-smooth muscle actin and extradomain A fibronectin but no significant increase of these proteins in OPN-null fibroblasts. Phalloidin staining for actin filaments and immunostaining for α-smooth muscle actin and focal adhesion proteins showed reduced stress fibers, focal adhesions, and lamellipodia in OPN-null fibroblasts compared with wild-type cells. OPN-null fibroblasts exhibited 40% to 60% less spreading, 50% less resistance to detachment by shear force, and a ≈3-fold reduction in collagen gel contraction. These defects were partially rescued by ectopic expression of OPN. Mass spectrometric analysis of proteins in focal adhesions formed on collagen type I beads revealed an enrichment of HMGB1 protein in wild-type cells, whereas HMGB1 was not detected in OPN-null cells. Treatment of wild-type cells with small interfering RNA to knock down OPN reduced transforming growth factor-β1–induced α-smooth muscle actin and HMGB1 to levels observed in OPN-null cells. These studies demonstrate that OPN is required for the differentiation and activity of myofibroblasts formed in response to the profibrotic cytokine transforming growth factor-β1.

Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin.

Expression of osteopontin (OPN) by activated T‐cells and macrophages is required for the development of cell‐mediated inflammatory responses. Acting through integrin αvβ3 and CD44 receptors, OPN can promote chemoattraction and pro‐inflammatory cytokine expression by macrophages. In this study, we have used periotoneal macrophages from OPN−/, CD44−/−, and WT mice to study the relationship between OPN and CD44 in macrophage migration. Using confocal microscopy, we show that OPN co‐distributes with CD44 inside macrophages at cell edges and in cell processes in a mutually dependent manner. The existence of an intracellular form of OPN is supported by pulse‐chase studies in which a thrombin‐sensitive, phosphorylated protein immunoprecipitated with OPN antibodies is retained inside macrophages. In OPN−/− and CD44−/− macrophages, the absence of CD44 and OPN, respectively, is associated with the formation of fewer cell processes, reduced cell fusion required to form functional multinucleated osteoclasts in the presence of CSF‐1 and RANKL, and impaired chemotaxis. Whereas the chemotaxis of CD44−/− cells to various chemoattractants is almost completely abrogated, a differential effect is seen with the OPN−/− cells. Thus, OPN−/− cells migrate normally towards CSF‐1 but not towards fMLP and MCP‐1, which signal through G‐protein coupled receptors (GPCRs). That the GPCR‐mediated migration is dependent upon the level of cell‐surface CD44 is indicated by the reduced cell‐surface expression of CD44 in OPN−/− cells and a comparable impairment in the chemotaxis of CD44+/− cells. Although chemotaxis of OPN−/− cells could be rescued by an OPN substratum, or by addition of high levels of OPN in solution, no response is evident with physiological levels of OPN, indicating a requirement for the CD44‐associated intracellular OPN in CD44 cell‐surface expression. These studies indicate, therefore, that the level of cell surface CD44 is critical for GPCR‐mediated chemotaxis by peritoneal macrophages and suggest that a novel intracellular form of OPN may modulate CD44 activities involved in these processes. J. Cell. Physiol. 198: 155–167, 2004.

Integrity of cell-cell contacts is a critical regulator of TGF-beta 1-induced epithelial-to-myofibroblast transition: role for beta-catenin.

Injury of the tubular epithelium and TGF-β1-induced conversion of epithelial cells to α-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-β1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-β1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca2+-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-β1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since β-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-β1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and β-catenin, but TGF-β1 selectively rescued β-catenin and stimulated the β-catenin-driven reporter TopFLASH. Moreover, chelation of free β-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-β1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a β-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-β1 are required for EMT.