Protein factors which regulate cell motility

Summary Cell motility (i.e., movement) is an essential component of normal development, inflammation, tissue repair, angiogenesis, and tumor invasion. Various molecules can affect the motility and positioning of mammalian cells, including peptide growth factors, (e.g., EGF, PDGF, TGF-beta), substrate-adhesion molecules (e.g., fibronectin, laminin), cell adhesion molecules (CAMs), and metalloproteinases. Recent studies have demonstrated a group of motility-stimulating proteins which do not appear to fit into any of the above categories. Examples include: 1)scatter factor (SF), a mesenchymal cell-derived protein which causes contiguous sheets of epithelium to separate into individual cells and stimulates the migration of epithelial as well as vascular endothelial cells; 2)autocrine motility factor (AMF), a tumor cell-derived protein which stimulates migration of the producer cells; and 3)migration-stimulating factor (MSF), a protein produced by fetal and cancer patient fibroblasts which stimulates penetration of three-dimensional collagen gels by non-producing adult fibroblasts. SF, AMF, and MSF are soluble and heat labile proteins with Mr of 77, 55, and 70 kd by SDS-PAGE, respectively, and may be members of a new class of cell-specific regulators of motility. Their physiologic functions have not been established, but available data suggest that they may be involved in fetal development and/or tissue repair.     

In vivo degradation of fetal wound hyaluronic acid results in increased fibroplasia, collagen deposition, and neovascularization.

Fetal tissue repair occurs without acute inflammation, prominent fibroplasia, or marked neovascularization. The fetal wound extracellular matrix is rich in hyaluronic acid (HA), while collagen is deposited in an organized normal dermal pattern. In various biologic systems, including regeneration and development, the controlled accumulation and subsequent degradation of hyaluronic acid is associated with distinct cellular and matrix events. Therefore, it is hypothesized that the abundance of hyaluronic acid in fetal wounds may influence cellular and/or matrix events such that tissue repair is highly organized and adult-like scarring does not occur. To test this hypothesis, the hyaluronic acid content of fetal rabbit wounds was reduced by specific degradation with Streptomyces hyaluronidase. Control wounds were treated with either enzyme buffer (n = 12) or denatured enzyme solution (n = 8) and exhibited a normal fetal healing response with scattered peripheral fibroblasts, a matrix of hyaluronic acid, and no infiltrating collagen. In marked contrast, the hyaluronidase-treated wounds (n = 14) demonstrated increased fibroblast infiltration, collagen deposition, and capillary formation. A significant reduction in the hyaluronic acid content of the hyaluronidase-treated wounds was confirmed biochemically. Since the degradation of hyaluronic acid resulted in an altered healing response, this study demonstrates that hyaluronic acid affects the cellular and matrix events in fetal healing and may be partially responsible for the unique qualities of this regenerative repair process.     

Co-expression by keratinocytes of migration stimulating factor (MSF) and a functional inhibitor of its bioactivity (MSFI)

Migration stimulating factor (MSF) is a potent autocrine and paracrine factor expressed by fibroblasts and epithelial cells in foetal skin, tumours and healing wounds. In tissue culture, MSF bioactivity is present in the conditioned medium of foetal and tumour derived fibroblasts, but not in normal adult fibroblasts or keratinocytes. The conditioned medium of early passage keratinocytes or a keratinocyte line (HaCaT) effectively inhibited the motogenic activity of rhMSF. Fractionation of keratinocyte conditioned medium by size-exclusion chromatography revealed the presence of bioactive MSF as well as a functional inhibitor of MSF (MSFI) in fractions corresponding to approximately 70 kDa and 25 kDa, respectively. MSFI was purified and identified as neutrophil gelatinase-associated lipocalin (NGAL or lipocalin-2). Immunostaining confirmed that keratinocytes expressed both MSF and NGAL, whereas normal adult fibroblasts did not express either. Recombinant and cell-produced NGAL neutralised the motogenic activity of rhMSF. NGAL is known to bind MMP-9 and promote the activity of this protease. In contrast, there was no evidence of NGAL-MSF binding in keratinocyte conditioned medium. MSF displays a number of bioactivities of relevance to cancer progression and wound healing. Our findings indicate a novel function of NGAL and a possible mechanism for regulating MSF activity in tissues.     

Differential Effects of TGF-β1 on Hyaluronan Synthesis by Fetal and Adult Skin Fibroblasts: Implications for Cell Migration and Wound Healing

Fetal wound healing differs from its adult counterpart in that it is regenerative and occurs without scarring. The matrix macromolecule hyaluronan (HA) and various cytokines, including members of the TGF-β family, have been implicated in the control of scarring. We have previously reported that adult and fetal fibroblasts differ with respect to the effect of cell density on HA synthesis when cultured on plastic tissue culture dishes. Data regarding the effects of substratum and TGF-β1 on HA synthesis by these cells are presented in this communication. Our results indicate that HA synthesis by both fetal and adult fibroblasts is (a) up-regulated by culture on a collagen substratum and (b) differentially regulated by TGF-β1 in a manner which is dependent upon both substratum and cell density. TGF-β1 stimulated HA synthesis by confluent fetal fibroblasts growing on a plastic substratum, but inhibited HA synthesis on a collagen substratum; these data underscore the important role of the substratum in determining the precise effect of TGF-β1 on cell behavior. Related studies indicated that the migration of fetal and adult fibroblasts into the collagen substrata was modulated by TGF-β1 in a manner identical to its effect on HA synthesis. These observations are discussed in terms of the contribution of distinct fibroblast subpopulations to wound healing and the manner in which this is regulated by matrix and cytokines.     

Heparin modulates the organization of hydrated collagen gels and inhibits gel contraction by fibroblasts

We studied the effects of extracellular matrix components on fibroblast contraction of hydrated collagen gels. After 4-h incubations, heparin-containing collagen gels contracted only 10% compared with 50% contraction of control gels. Contraction was not affected by hyaluronic acid, dermatan sulfate, or fibronectin, implying that the activity of heparin was specific. The possibility that heparin inhibited attachment of the cells to the gels was ruled out. Also, addition of heparin to the incubation medium had no effect on contraction. Microscopic examination showed that control collagen gels were composed of a uniform network of interlocking fibrils of similar sizes. Heparin-containing gels, on the other hand, were highly variable with some collagen bundles containing 5-6 collagen fibrils and other regions containing amorphous material. Unlike the control gels, the fibrils of heparin-containing gels were not continuously interconnected. Based on the results, we propose that fibroblasts attach normally to the collagen fibrils of heparin-containing gels and attempt to contract the gels, but the mechanical forces exerted by fibroblasts on individual collagen fibrils cannot be propagated throughout the gels.     

Contraction of fibrillar type I collagen by endothelial cells: A study in vitro

The formation of microvascular sprouts during angiogenesis requires that endothelial cells move through an extracellular matrix. Endothelial cells that migrate in vitro generate forces of traction that compress (i.e., contract) and reorganize vicinial extracellular matrix, a process that might be important for angiogenic invasion and morphogenesis in vivo. To study potential relationships between traction and angiogenesis, we have measured the contraction of fibrillar type I collagen gels by endothelial cells in vitro. We found that the capacity of bovine aortic endothelial (BAE) cells to remodel type I collagen was similar to that of human dermal fibroblasts—a cell type that generates high levels of traction. Contraction of collagen by BAE cells was stimulated by fetal bovine serum, human plasma-derived serum, bovine serum albumin, and the angiogenic factors phorbol myristate acetate and basic fibroblast growth factor (bFGF). In contrast, fibronectin and immunoglobulin from bovine serum, several nonserum proteins, and polyvinyl pyrrolidone (a nonproteinaceous substitute for albumin in artificial plasma) were not stimulatory. Contraction of collagen by BAE cells was diminished by an inhibitor of metalloproteinases (1, 10-phenanthroline) at concentrations that were not obviously cytotoxic. Zymography of proteins secreted by BAE cells that had contracted collagen gels revealed matrix metalloproteinase 2. Subconfluent BAE cells that were migratory and proliferating were more effective contractors of collagen than were quiescent, confluent cells of the same strain. Moreover, bovine capillary endothelial cells contracted collagen gels to a greater degree than was seen with BAE cells. Collectively, our observations indicate that traction-driven reorganization of fibrillar type I collagen by endothelial cells is sensitive to different mediators, some of which, e.g., bFGF, are known regulators of angiogenesis in vivo. © 1996 Wiley-Liss, Inc.     

Migration Stimulating Factor (MSF) promotes fibroblast migration by inhibiting AKT

The protein kinase AKT is activated strongly by many motogenic growth factors, yet has recently been shown capable of inhibiting migration in several cell types. Here we report that treatment with Migration Stimulating Factor (MSF), a truncated form of fibronectin that promotes the migration of many cell types, inhibits AKT activity in human fibroblasts and endothelial cells. In fibroblasts, treatment with either MSF or the AKT inhibitor, Akti-1/2, stimulated migration into 3D collagen gels to a similar extent and the effects of Akti-1/2 on migration could be blocked by the expression of an inhibitor-resistant mutant, AKT1 W80A. These data indicate that MSF promotes fibroblast migration, at least in part, by inhibiting the activity of AKT.     

Influence of serum on adult and fetal dermal fibroblast migration adhesion, and collagen expression

Summary The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater faction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.     

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.     

Motogenic and biosynthetic response of adult skin fibroblasts to TGF-beta isoforms (-1, -2 and -3) determined by 'tissue response unit': role of cell density and substratum

We have recently demonstrated that the three principal mammalian isoforms of transforming growth factor beta (TGF-beta) exert distinct effects upon: (1) the migration of confluent adult fibroblasts into 3D gels of native type I collagen fibres (i.e. TGF-beta-1 and -2 had no apparent motogenic activity, whilst TGF-beta-3 induced a dose-dependent stimulation of cell migration); and (2) the synthesis of hyaluronan (HA) by these cells is also affected by the TGF-beta isoforms in a manner which parallels their effect on cell migration. The objective of the present study is to elucidate the manner in which this differential activity of the TGF-beta-1, -2 and -3 may be modulated by experimental parameters. Data presented in this communication indicate that cytokine bioactivity is determined by a combination of cell density and the nature of the macromolecular substratum. Thus, we now report that all three TGF-beta isoforms inhibit the migration of subconfluent cells in the collagen gel assay. Our data confirm that the migration of confluent cells is stimulated by TGF-beta-3 and further indicate that this motogenic activity is completely abrogated by either TGF-beta-1 or -2 when these are co-incubated with TGF-beta-3. In contrast to these results obtained using a native type I collagen substratum, all three isoforms stimulated adult fibroblast migration in the transmembrane assay (in which cells are adherent to a 2-D porous polycarbonate substratum). The precise effect of TGF-beta isoforms on HA synthesis was also affected by cell density and the nature of the substratum in a manner which paralleled their diverse effects on cell migration (i.e. stimulation, inhibition or no effect). Streptomyces hyaluronidase completely neutralized the TGF-beta-3-induced stimulation of confluent fibroblast migration, thus suggesting a mechanistic link between the cytokine-induced cell migration and HA synthesis under these conditions. Taken together, these data indicate that: (1) the bioactivity of TGF-beta-1, -2 and -3 are determined by cell density, the macromolecular substratum and the presence of other cytokines; and (2) it is therefore necessary to define cytokine bioactivity within the context of a larger 'tissue response unit' which more fully defines the activity state of the target cell and its microenvironment.     

Interaction of human breast fibroblasts with collagen I increases secretion of procathepsin B

Interactions of stromal and tumor cells with the extracellular matrix may regulate expression of proteases including the lysosomal proteases cathepsins B and D. In the present study, we determined whether the expression of these two proteases in human breast fibroblasts was modulated by interactions with the extracellular matrix component, collagen I. Breast fibroblasts were isolated from non-malignant breast tissue as well as from tissue surrounding malignant human breast tumors. Growth of these fibroblasts on collagen I gels affected cell morphology, but not the intracellular localization of vesicles staining for cathepsin B or D. Cathepsins B and D levels (mRNA or intracellular protein) were not affected in fibroblasts growing on collagen I gels or plastic, nor was cathepsin D secreted from these cells. In contrast, protein expression and secretion of cathepsin B, primarily procathepsin B, was induced by growth on collagen I gels. The induced secretion appeared to be mediated by integrins binding to collagen I, as inhibitory antibodies against alpha(1), alpha(2), and beta(1) integrin subunits prevented procathepsin B secretion from fibroblasts grown on collagen. In addition, procathepsin B secretion was induced when cells were plated on beta(1) integrin antibodies. To our knowledge, this is the first examination of cathepsin B and D expression and localization in human breast fibroblasts and their regulation by a matrix protein. Secretion of the cysteine protease procathepsin B from breast fibroblasts may have physiological and pathological consequences, as proteases are required for normal development and for lactation of the mammary gland, yet can also initiate and accelerate the progression of breast cancer.     

Regeneration of corneal tissue

Penetrating wounds in rabbit corneas heal to form an opaque tissue that eventually becomes transparent. DNA content, dry weight, water content, and collagen content of the tissue gradually become more like that of normal cornea. The healing tissues also synthesize low-sulfated keratan sulfate, hyaluronic acid, and heparan sulfate. These glycosaminoglycans are not found in normal adult corneas but have been reported in fetal corneas. Previous studies have shown that collagen from healing corneal wounds and collagen from fetal corneas have very similar cross-linking patterns, but these patterns are different from those in normal adult collagen. The similarities between collagen and glycosaminoglycans in healing corneal wounds and in fetuses suggest some recapitulation of ontogenetic processes. The biochemical sequence and eventual return of transparency to the rabbit cornea indicate a capability for true regeneration of stromal tissue in the rabbit.     

Persistence of TGF-β1 induction of increased fibroblast contractility

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.     

HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.     

The behaviour of BHK cells and neutrophil leukocytes on collagen gels of defined mechanical strength

This study demonstrates how the mechanical strength of a series of collagen/composite gels can be measured using a penetrometer. It was found that the presence of fibrin in collagen gels resulted in increased gel strength. Similarly hyaluronic acid was found to increase the strength of collagen gels. Addition of heparin weakened collagen gels as did chondroitin-6-sulphate. Neutrophil migration into collagen gels was found to be inversely proportional to gel strength. Fibrin and hyaluronic acid containing gels inhibited neutrophil migration while the presence of heparin and chondroitin sulphate increased neutrophil migration. BHK gel contraction experiments demonstrated how the presence of fibrin prevents gel contraction. Despite increasing gel strength the presence of hyaluronic acid appeared to have no effect on BHK contraction of collagen gels. Similarly the presence of heparin or chondroitin sulphate had no effect on gel contraction by BHK cells.     

Effects of adenosine 3'':5''-cyclic monophosphate and serum on synthesis of hyaluronic acid in confluent rat fibroblasts.

A small amount of hyaluronic acid is synthesized in confluent cultures of rat fibroblasts, which have a high content of cyclic AMP. Addition of calf serum caused a rapid decrease in the cellular cyclic AMP content and large increases in hyaluronic acid synthetase activity and hyaluronic acid production. Addition of cyclic AMP also caused a marked increase in hyaluronic acid synthetase activity within 2h and then increased hyaluronic acid production. The effects of cyclic AMP and serum on hyaluronic acid synthesis were additive. Prostaglandin E2, which increased the cyclic AMP by stimulating adenylate cyclase, was as effective as cyclic AMP in increasing hyaluronic acid synthetase activity, but AMP was far less effective than cyclic AMP. These results indicate that cyclic AMP itself stimulates the mucopolysaccharide synthesis and that the effect of serum is not due to a decrease in cyclic AMP in the cells.     

The Effects of Age and the Expression of SPARC on Extracellular Matrix Production by Cardiac Fibroblasts in 3-D Cultures

Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts.     

Fetal wound healing: a biochemical study of scarless healing

Human fetal surgery is being successfully performed today in a small number of highly selected patients for conditions that may lead to irreversible damage to the fetus and threaten the viability of the newborn. Following surgical repair, fetal wounds heal without scarring. This study was initiated to characterize fetal wounds both histologically and biochemically. Gore-Tex tubing was implanted into the subcutaneous tissue of the back of fetal, newborn, and adult New Zealand white rabbits. Light microscopic examination of healed wounds revealed no evidence of scar formation. Electron microscopy demonstrated a striated fibrillar structure suggestive of collagen within the lumen of the Gore-Tex tubing implants. Amino acid analysis (sensitivity 40 pmol) confirmed the presence of hydroxylysine and hydroxyproline within the Gore-Tex wound chambers indicating the presence of collagen in fetal wounds. The small amount of collagen precluded the typing of the collagen using cyanogen bromide peptide analysis. The absence of scarring and the small amounts of detectable collagen suggest a high degree of reorganization of the connective tissues involved in repair. The fetal wound matrix is rich in hyaluronic acid. Topical hyaluronic acid has been associated experimentally with a reduced amount of scarring in postnatal wound healing. Hyaluronic acid extracted from human skin and scar tissue is associated with collagen and other proteins. We propose that a hyaluronic acid-collagen-protein complex may play a role in fetal wound healing.