Recombinant human platelet‐derived growth factor and transforming growth factor‐β mediated contraction of human dermal fibroblast populated lattices is inhibited by Rho/GTPase inhibitor but does not require phosphatidylinositol‐3′ kinase

Abstract

Matrix reorganization and tissue contraction are essential for wound healing. However, the intracellular signals that mediate these processes are largely unknown. We investigated cytokine-induced signaling and its potential role in contraction of adult human dermal fibroblast populated collagen lattices. The results document that recombinant human platelet-derived growth factor-BB and transforming growth factor-1 individually stimulate contraction of fibroblast populated collagen lattices, while a combination of the two cytokines leads to increased contraction. Although recombinant human platelet-derived growth factor-BB promoted collagen contraction, it failed to stimulate phosphatidylinositol-3' kinase in the human dermal fibroblasts. An inhibitor for phosphatidylinositol 3' kinase, wortmannin, had no effect on the cytokine-mediated collagen contraction. In addition, this failed activation of phosphatidylinositol 3' kinase is consistent with absence of tyrosine phosphorylation of the platelet-derived growth factor receptor when the cells are in a collagen matrix. In contrast, tyrosine phosphorylation of the platelet-derived growth factor receptor was readily detected in the dermal fibroblasts in monolayers. We also probed the potential role of Rho/GTPase in the cytokine-mediated contraction of fibroblast populated collagen lattices. Toxin B from Clostridium difficile at picomolar concentrations blocked both recombinant human platelet-derived growth factor and transforming growth factor-5 induced contraction. Further, this inhibition was correlated with the inhibition of cell spreading in collagen, which suggests the formation of actin fibers inside the cells is essential for cytokine-mediated contraction of fibroblast populated collagen lattices. Taken together, these results imply that Rho/GTPase signaling but not phosphoinositol-3' kinase is involved in the cytokine-mediated contraction of fibroblasts populated collagen lattice. These findings suggest a potential mechanism for these signaling components during human wound contraction.

The effect of the amelogenin fraction of enamel matrix proteins on fibroblast-mediated collagen matrix reorganization

“…Exogenously added TGF-b1 accelerated DE contraction in our study in agreement with numerous previous reports [24][25][26][27][28][29][30]. TGF-b1 is thought to regulate DE contraction by altering the composition and tertiary structure of the ECM as well as integrin expression [28,41] but without influencing DNA synthesis in the fibroblasts [42].…”

“…The PDGF-AA and PDGF-BB isoforms caused more rapid contraction than did TGF-b1 in a similar DE system to ours [24]. Furthermore, another group showed that the effect of PDGF-BB and TGF-b1 on DE contraction was additive [27] through independent signalling pathways [24]. This is a plausible explanation for the significantly higher response of both EMP and amelogenin than TGF-b1 treatment alone.…”

“…TGF-b1 is known to promote DE contraction [24][25][26][27][28][29][30] and TGF-b1 levels were therefore determined by a specific ELISA assay in conditioned media from the different groups as a measure of TGF-b1 production by the fibroblasts.…”

“…Because it is unclear whether the effect of EMP can be ascribed to amelogenin, a comparison between EMP and recombinant porcine amelogenin, devoid of TGF-b1 or other growth factors, was also made. Furthermore, TGF-b1 stimulates remodelling of the DE [24][25][26][27][28][29][30] and was used as a positive control in the studies.…”

Modulation of Corneal Fibroblast Contractility within Fibrillar Collagen Matrices

“…Consistent with these data, we observed rapid pseudopodial and/or filopodial extension coincident with cell relaxation after Rho-kinase inhibition, suggesting a relative increase in Rac activity. Others have demonstrated that Rho or Rho-kinase inhibition decreases the overall contraction of collagen matrices, 45,63,89 reduces force generation by a mass culture of cells, 90 and blocks LPA-induced retraction of dendritic cell processes in floating collagen matrices. 91 However, to our knowledge, this is the first study to demonstrate directly the effect of Rho-kinase inhibition on the subcellular pattern of force generation by individual cells in a 3-D model.…”