Cell coupling modulates the contraction of fibroblast-populated collagen lattices

Cultured dermal fibroblasts become notably elongated when incorporated into a fibroblast-populated collagen lattice (FPCL). With time these fibroblasts reorganize the collagen responsible for reduction in lattice size. In monolayer the microinjection of Lucifer Yellow (LY) into cultured human fibroblasts shows cell coupling through gap junctions. Human fibroblasts residing on the periphery of a FPCL are at high density and the microinjection of LY into one of those fibroblasts demonstrates cell coupling. Cells within the center of an FPCL are at low density and appear to be independent of one another; however, the microinjection of LY into selected fibroblasts again demonstrates cell coupling. Hence the microinjection of cells in both the center and the edge of a FPCL pass dye to numerous neighbors. Does cell coupling influence FPCL contraction? FPCL incubated with heptanol and octanol, aliphatic alcohols that uncouple cells, inhibits lattice contraction, whereas hexanol, an aliphatic alcohol that does not uncouple cells, did not alter lattice contraction. Fibroblasts derived from connexin 43 (a transmembrane protein responsible for gap junction structures) knockout mice were demonstrated to lack gap junctional communications. When incorporated into a FPCL these cells failed to elongate and demonstrated retarded lattice contraction. Hence, gap junctional communications between fibroblasts incorporated into collagen lattices appear to optimize FPCL contraction and suggest a role for gap junctions in the organization of collagen fibers.     

Cell locomotion forces versus cell contraction forces for collagen lattice contraction: an in vitro model of wound contraction

Cultured human dermal fibroblasts suspended in a rapidly polymerizing collagen matrix produce a fibroblast-populated collagen lattice. With time, this lattice will undergo a reduction in size referred to as lattice contraction. During this process, two distinct cell populations develop. At the periphery of the lattice, highly oriented sheets of cells, morphologically identifiable as myofibroblasts, show cell-to-cell contacts and thick, actin-rich staining cytoplasmic stress fibers. It is proposed that these cells undergoing cell contraction produce a multicellular contractile unit which reorients the collagen fibrils associated with them. The cells in the central region, referred to as fibroblasts, are randomly oriented, with few cell-to-cell contacts and faintly staining actin cytoplasmic filaments. In contrast it is proposed that cells working as single units use cell locomotion forces to reorient the collagen fibrils associated with them. Using this model, we sought to determine which of these two mechanisms, cell contraction or cell locomotion, is responsible for the force that contracts collagen lattices. Our experiments showed that fibroblasts produce this contractile force, and that the mechanism for lattice contraction appears to be related to cell locomotion. This is in contrast to a myofibroblast; where the mechanism for contraction is based upon cell contractions. Fibroblasts attempting to move within the collagen matrix reorganize the surrounding collagen fibrils; when these collagen fibrils can be organized no further and cell-to-cell contacts develop, which occurs at the periphery of the lattice first, these cells can no longer participate in the dynamic aspects of lattice contraction.     

Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.     

The effect of myofibroblast on contracture of hypertrophic scar

Wound contraction in humans has both positive and negative effects. It is beneficial to wound healing by narrowing the wound margins, but the formation of undesirable scar contracture brings cosmetic and even functional problems. The entire mechanism of wound healing and scar contracture is not clear yet, but it is at least considered that both the fibroblasts and the myofibroblasts are responsible for contraction in healing wounds. The myofibroblast is a cell that possesses all the morphologic and biochemical characteristics of both a fibroblast and a smooth muscle cell. Normally, the myofibroblasts appear in the initial wound healing processes and generate contractile forces to pull both edges of an open wound until it disappears by apoptosis. But as an altered regulation of myofibroblast disappearance, they remain in the dermis and continuously contract the scar, eventually causing scar contracture. In this research, to compare and directly evaluate the influence on scar contracture of the myofibroblast versus the fibroblast, dermal tissues were taken from 10 patients who had highly contracted hypertrophic scars. The myofibroblasts were isolated and concentrated from the fibroblasts using the magnetic activating cell-sorting column to obtain the myofibroblast group, which contained about 28 to 41 percent of the myofibroblasts, and the fibroblast group, which contained less than 0.9 percent of the myofibroblasts. Each group was cultured in the fibroblast-populated collagen lattice for 13 days, and the contraction of the collagen gel was measured every other day. In addition, they were selectively treated with tranilast [N-(3',4'-dimethoxycinnamoyl) anthranilic acid] to evaluate the influence on the contraction of the collagen gel lattice. During the culture, the myofibroblast group, compared with the fibroblast group, showed statistically significant contraction of the collagen gel lattice day by day, except on the first day, and only the myofibroblast group was affected by tranilast treatment, showing significant inhibition of gel contraction. By utilizing an in vitro model, the authors have demonstrated that myofibroblasts play a more important role in the contracture of the hypertrophic scar.     

Dupuytren's disease: physiologic changes in nodule and cord fibroblasts through aging in vitro

The pathogenesis of the fibrotic disease Dupuytren's contracture remains unclear. The disease process includes two structurally distinct fibrotic elements, the nodule and the cord. It has been proposed that as the disease progresses, nodules develop into cords. To corroborate that hypothesis, the authors took advantage of cultured fibroblast differences found between gap junction intercellular communication and fibroblast-populated collagen lattice contraction. Paired fibroblast cell lines of nodules and cords derived from four patients with Dupuytren's disease were maintained in culture for at least eight passages. The presence of gap junction intercellular communication in nodule- and cord-derived fibroblasts was documented and reported as a coupling index. The contraction of free-floating nodule- or cord-derived collagen lattices was also documented and reported. Early passage (passage 4) cord-derived fibroblasts showed a significant increase in coupling index compared with passage 4 nodule-derived fibroblasts (4.0 +/- 0.4 versus 2.5 +/- 0.3, respectively), where p < or = 0.01. However, late passage (passage 8) nodule- and cord-derived fibroblasts were equivalent in their coupling index (4.1 +/- 0.4 versus 4.4 +/- 0.4, respectively). Early passage nodule-derived fibroblast-populated collagen lattices contracted by 64 percent, whereas late passage nodule-derived lattices showed less contraction, at only 40 percent. Early and late passage cord-derived lattices contracted 46 and 37 percent, respectively. All nodule- and cord-derived cell lines were statistically equivalent at lattice contraction by passage 8. These in vitro studies support the hypothesis that fibroblasts derived from Dupuytren's contracture nodules change their phenotype after undergoing repeated cell passage, acquiring a cord-like fibroblast phenotype. Dupuytren's nodules represent the early, active form of fibrosis in which cells are more proliferative, better at fibroblast-populated collagen lattice contraction, and display less gap junction intercellular communication. The speculation is that alterations in gap junction intercellular communication may be involved in the progression of Dupuytren's nodules to cords as the disease progresses.     

Role of vitronectin and fibronectin receptors in oral mucosal and dermal myofibroblast differentiation

Background information. The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient‐matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts). Results. Both the HOF and HDF cell types underwent TGF‐β1 (transforming growth factor‐β1)‐induced myofibroblastic differentiation [upregulation of the expression of α‐sma (α‐smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of α‐sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits α5 (fibronectin) or αv (vitronectin) were used to determine whether the effects of TGF‐β1 were regulated via integrin signalling pathways. α‐sma expression in both HOFs and HDFs was down‐regulated by antibodies against both α5 and αv. Functionally, TGF‐β1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF‐β1 (P<0.05). When TGF‐β1‐stimulated cells were incubated with blocking antibodies against α5 and αv, gel contraction was decreased to that of non‐stimulated cells; however, blocking αv or α5 could not restore cellular migration in both HOFs and HDFs. Conclusions. Despite intrinsic differences in their basal state, the cellular events associated with TGF‐β1‐induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up‐regulation of α‐sma expression and increases in collagen gel contraction are vitronectin‐ and fibronectin‐receptor‐dependent processes, whereas wound re‐population is not.     

Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.     

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL-10 is a potent anti-inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL-10 alleviates lipopolysaccharide (LPS)-induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS-derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS-derived fibroblasts (NSFs). LPS could up-regulate the expression of TLR4, pp65, Col I, Col III and α-SMA in NSFs, but IL-10 could down-regulate their expression in both HSFs and LPS-induced NSFs. Blocking IL-10 receptor (IL-10R) or the phosphorylation of STAT3, their expression was up-regulated. In addition, in vitro and in vivo models results showed that IL-10 could alleviate LPS-induced fibroblast-populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL-10 alleviates LPS-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL-10 can alleviate the LPS-induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down-regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.     

Differences in the mechanism for high- versus moderate-density fibroblast-populated collagen lattice contraction

The free-floating fibroblast-populated collagen lattice (FPCL) model introduced by Bell contains 0.5 x 10(5) cell/ml and here is defined as a moderate-density FPCL (MD-FPCL). One modification of the model is to increase the cell density by a factor of 10, where 5 x 10(5) cells/ml defines a high-density FPCL (HD-FPCL). The initial detection of HD-FPCL contraction is 2 h, whereas MD-FPCL is later, 6 h. A contracted HD-FPCL has a doughnut-like appearance, due to the high density of cells accumulating at the periphery. A contracted MD-FPCL is a flattened disc. The compacted collagen of MD-FPCL lattice exhibits a strong birefringence pattern due to organized collagen fiber bundles. In contracted HD-FPCL, a minimal birefringence develops, indicating minimal organization of collagen fiber bundles. MD-FPCL contraction was reduced with less than 10% serum; the disruption of microtubules, uncoupling of gap junctions, inhibition of tyrosine kinases, and addition of a blocking antibody to alpha2beta1 collagen integrin. Making HD-FPCL with only 1% serum or including the inhibitory agents had only minimal affect on lattice contraction. On the other hand, platelet-derived growth factor stimulated HD-FPCL contraction but had no influence on MD-FPCL contraction. It is suggested that the mechanism for HD-FPCL contraction is limited to the process of cells spreading. HD-FPCL contraction is independent of collagen organization, microtubules, gap junctions, alpha2beta1 integrin, and tyrosine phosphorylation. MD-FPCL contraction involves collagen organization and is optimized by the involvement of microtubules, gap junctions, alpha2beta1 integrin, and tyrosine phosphorylation. When studying cell physiology in a collagen matrix, cell-density influences need to be considered.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down- regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine- specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.     

Mechanical behavior of fibroblasts included in collagen lattices

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin. Excessive steroid activity, genetic and mechanical factors and inherited defects of connective tissues are the most frequent causes of this disease. Fibroblasts derived from women presenting striae distensae lesions were included into collagen gels to study their mechanical behavior: capacity to contract free-floating lattices and to produce isometric force in tense lattices. To measure the retracted lattice diameter, the culture dishes were placed on a transparent metric scale. An isometric force system was used to study quantitatively the forces developed during lattice contraction. alpha 2 beta 1 integrins expression (transmembrane receptors) was evaluated by flux cytometry. Striae distensae fibroblasts contract collagen gels slower than normal human fibroblasts but the final contraction is similar. They produce a greater isometric force which is associated with enhanced alpha 2 beta 1 integrins expression. By their mechanical properties, striae distensae fibroblasts appear as a different population from normal fibroblasts.     

Gamma radiation inhibits fibroblast-mediated collagen gel retraction

Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role in the production and modification of extracellular matrix in wound repair. Since one important aspect of wound repair is the contraction of the wound, this study investigated the effects of radiation on the ability of fibroblasts to mediate collagen gel contraction in an in vitro model of wound retraction. After irradiation, the cells were detached and suspended in a solution of rat tail tendon collagen. Radiation exposure decreased retraction, and this effect was dose dependent. In order to define the mechanism of reduced gel retraction, we investigated alpha2beta1 cell surface integrin and fibronectin, which are thought to mediate contraction, and prostaglandin E2 (PGE2), which is known to inhibit this process. PGE2 release increased dose responsively following radiation. The cyclooxygenase inhibitor indomethacin could partially restore the contractile activity of irradiated fibroblasts. Fibronectin production in gel culture showed a significant decrease. In contrast, there was no decrease in alpha2beta1 integrin expression in radiated cells. In conclusion, radiation decreases fibroblast-mediated gel contraction. Increased PGE2 production and decreased fibronectin production by irradiated fibroblasts may contribute to this effect and may be in part responsible for poor healing of radiated tissue.     

Platelet-derived growth factor and inflammatory cytokines have differential effects on the expression of integrins α1β1 and α5β1 by human dermal fibroblasts in vitro

Dermal fibroblasts are essential for the repair of cutaneous wounds. Fibroblasts presumably use cell surface receptors of the integrin family during migration into a wound from the adjacent uninjured tissue and for the subsequent matrix repairs. We have investigated the possible roles of platelet-derived growth factor and inflammatory cytokines in the regulation of integrin expression on wound fibroblasts using a porcine cutaneous wound model and cultured human cells. Tissue specimens collected from 4-day pig wounds were stained with antibodies specific for the α1 and α5 integrin subunits. Staining for α1 was markedly decreased on fibroblasts adjacent to the wound and in the granulation tissue, while staining for α5 was clearly enhanced in both locations. Normal adult human dermal fibroblasts in culture express the integrins α1β1, a collagen receptor, and α5β1, a fibronectin receptor. Quantitative flow cytometry was used to measure cell surface integrin expression after treatment with platelet-derived growth factor (PDGF)-AA, PDGF-AB, or PDGF-BB. Each isoform of PDGF produced a significant decrease in the level of α1 present on the cell surface and an increase in the level of α5. Furthermore, PDGF-BB produced a corresponding decrease in α1 mRNA and an increase in α5 mRNA. In contrast, treatment with three inflammatory cytokines, IL-1β, TNF-α, and IFN-γ, produced clear increases in the levels of α1 and α5 present on the cell surface. Our observations suggest that the differential effects of PDGF and inflammatory cytokines may be part of the mechanism regulating the expression of α1 and α5 integrins by dermal fibroblasts during wound repair. © 1996 Wiley-Liss, Inc.     

Fetal and adult human skin fibroblasts display intrinsic differences in contractile capacity.

One of the differences between fetal and adult skin healing is the unique ability of fetal wounds to heal without contracture and scar formation. Studies have shown that the ratio between the three isoforms of TGFbeta is different in adult and fetal wounds. Thus, we analyzed the capacity of adult and fetal human skin fibroblasts to contract collagen gels after stimulation with TGFbeta isoforms. In control medium, fetal fibroblasts had a contractile capacity similar to that of adult fibroblasts. However, the growth capacity of fetal fibroblasts was completely inhibited, in contrast to adult fibroblasts. When cells were treated with TGFbeta, fetal fibroblasts showed an inhibition of their contractile capacity whereas adult fibroblasts further contracted gels. The contractile response was similar for all isoforms of TGFbeta although TGFbeta3 always had the strongest effect. We considered that the regulation of cell contractile capacity by TGFbeta may be dependent on receptor expression for this cytokine, on myofibroblast differentiation of the cells, or in cell links with matrix. Since TGFbeta receptor analysis did not show differences in receptor affinity, we studied the expression of alpha-smooth muscle (SM) actin, a fibroblast contractile marker and of three integrins, the cell surface receptors specific of the attachment of the fibroblasts with collagen matrix. We observed that the expression of alpha-SM actin and alpha3 and beta1 integrin subunits was increased when TGFbeta was added to the medium of adult fibroblasts whereas the levels of the alpha1 and alpha2 subunits were unchanged. In contrast, fetal fibroblasts treated with TGFbeta showed a decrease of alpha1, alpha2, and beta1 integrin expression but no change in alpha3 integrin and in alpha-SM actin expression. These results indicate that intrinsic differences between fetal and adult fibroblasts might explain their opposite responses to TGFbeta stimuli. The variations in their alpha-SM actin and integrin expression patterns represent potentially important mechanisms used by fetal fibroblasts to regulate their response to cytokines, and likely contribute to the resultant differences in the quality of wound repair.     

Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells

The capacity of cells to organize and contract collagen fibrils is fundamental to processes as diverse as embryogenesis and wound healing. We analyzed different beta 1 integrins on diploid fibroblasts for their role in modifying the tertiary structure of collagen matrices. Using monoclonal antibodies that block the interaction of integrins with their ligands, evidence was obtained that alpha 2 beta 1 integrin is required for the contraction of a type I collagen matrix. Further supporting the role of alpha 2 beta 1, cell lines expressing minimal levels of this integrin uniformly failed to contract collagen matrices. In addition, transfection of a full-length alpha 2 cDNA into one such cell line led to enhanced cell surface expression of alpha 2 beta 1 and conferred the de novo capacity to contract collagen matrices.     

The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: implications for wound healing

Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin alpha3beta1- and/or alpha6beta4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-beta1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Receptor tyrosine kinase signaling required for integrin alpha v beta 5- directed cell motility but not adhesion on vitronectin

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.