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PubMed1995

Identification of integrin alpha 3 beta 1 as a neuronal thrombospondin receptor mediating neurite outgrowth.

Thrombospondins are a family of extracellular matrix proteins expressed throughout the developing nervous system that promote neurite outgrowth in vitro and help mediate the migration of granule cells across the molecular layer in explants of neonatal cerebellum. The receptors mediating these interactions have not previously been identified. In this study, monoclonal antibodies raised to the integrin alpha 3 beta 1 heterodimer are shown to inhibit neurite outgrowth by rat sympathetic neurons on thrombospondin-1. Alpha 3 beta 1 is found to be expressed on the cell body, neurites, and growth cones of sympathetic neurons in vitro and on sympathetic axons passing through the thrombospondin-rich outer sheath of the superior cervical ganglion in vivo, consistent with its role in mediating axon outgrowth. A receptor-ligand binding assay is used to demonstrate the direct binding of immunopurified alpha 3 beta 1 to thrombospondin-1. These results demonstrate a direct interaction between the integrin alpha 3 beta 1 and thrombospondin-1, which mediates neurite outgrowth in vitro and is likely to mediate the same interactions in vivo.

Adrenergic Fibers↗
PubMed2006

siRNA targeted against HAb18G/CD147 inhibits MMP-2 secretion, actin and FAK expression in hepatocellular carcinoma cell line via ERK1/2 pathway.

HAb18G/CD147 has been identified as a factor that induces MMPs production. SiRNA targeted against HAb18G/CD147 was transfected into FHCC-98 cells (a HCC cell line) to knockdown its expression. The results showed that downregulating HAb18G/CD147 decreased ERK1/2, MMP-2 and FAK levels and inhibited cell motility and invasion, together with rearranged actin stress fiber formation, while had no effects on integrin alpha3beta1 expression. MEK1/2 inhibitor, U0126, inhibited MMP-2, FAK and actin expression in FHCC-98 cell line. The findings indicate that si-HAb18G inhibits gelatinase production, actin and FAK expression in FHCC-98 via an ERK1/2 signaling pathway.

Actins↗
PubMed2005

Podosomes as smart regulators of cellular adhesion.

Podosomes are punctate adhesion structures first described in osteoclasts and next found in src-transformed cells of mesenchymal origin. Podosomes were never observed in cultured epithelial cells where cell-matrix adhesion structures were represented only by focal contacts and hemidesmosomes interacting with microfilaments and intermediate filaments, respectively. Rat bladder carcinoma cells and normal human keratinocytes showed that hemidesmosome-like structures are organized around a core of actin filaments that appears early during cell adhesion and looks similar to those of podosomes described in cells of mesenchymal origin. The epithelial podosome-like structures specifically contain Arp2/3 complex, cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin and zyxin. The maintenance of the F-actin core and the surrounding hemidesmosomes depends on actin polymerization, src family kinases and Grb2, but not on microtubular integrity. Thus, podosomes are not unique to cells of mesenchymal origin, but also appear in epithelial cells where they may take part in regulating basement membrane adhesion.

Actin Cytoskeleton↗
PubMed2004

Keratinocytes from patients lacking collagen XVII display a migratory phenotype.

Acquired or inherited junctional epidermolysis bullosa are skin diseases characterized by a separation between the epidermis and the dermis. In inherited nonlethal junctional epidermolysis bullosa, genetic analysis has identified mutations in the COL17A1 gene coding for the transmembrane collagen XVII whereas patients with acquired diseases have autoantibodies against this protein. This suggests that collagen XVII participates in the adhesion of basal keratinocytes to the extracellular matrix. To test this hypothesis, we studied the behavior of keratinocytes with null mutations in the COL17A1 gene. Initial adhesion of mutant cells to laminin 5 was comparable to controls and similarly dependent on alpha3beta1 integrins. The spreading of mutant cells was, however, enhanced, suggesting a propensity to migrate, which was confirmed by migration assays. In addition, laminin 5 deposited by collagen XVII-deficient keratinocytes was scattered and poorly organized, suggesting that correct integration of laminin 5 within the matrix requires collagen XVII. This assumption was supported by the co-distribution of the two proteins in the matrix of normal human keratinocytes and by protein-protein-binding assays showing that the C-terminus of collagen XVII binds to laminin 5. Together, the results unravel an unexpected role of collagen XVII in the regulation of keratinocyte migration.

Autoantigens↗
PubMed2000

Expression of alpha and beta integrins during terminal differentiation of cardiomyocytes.

BACKGROUND: In the myocardium, myocyte cell division is irreversibly blocked shortly after birth. The signal that initiates cell cycle withdrawal is unknown. The purpose of this study was to relate changes in expression of beta1 integrin and its associated alpha subunits to cardiomyocyte cell cycle progression during the fetal-to-neonatal developmental transition in rat. METHODS AND RESULTS: The developmental expression pattern and function of beta 1 integrin and several of its associated alpha subunits were examined using reverse transcription (RT) polymerase chain reaction (PCR) and beta 1 blocking antibodies. During the fetal to neonatal transition, a dramatic shift occurred in the levels of beta1 and alpha isoforms. At the 17-day fetal stage only beta 1A was present, which remained relatively constant until immediately after birth then decreased by 30% at the adult stage. By contrast, beta 1D appeared at fetal day 18, increased at neonatal day 2, and afterwards remained constant. This resulted in a ratio of beta 1A to beta 1D of about 1:1 in the adult heart. The integrin beta 1-associated subunits, alpha 3, alpha 6, and alpha 7, were expressed at extremely low levels in 17-day fetal cardiomyocytes. After birth alpha 3 and alpha 6 transiently increased at the 2-day neonatal stage, while alpha 7 isoforms B, C, and X2 progressively increased to the adult stage. Unlike skeletal muscle cells, fluorescence-activated cell sorting analysis (FACS) showed no down regulation of the alpha 5 beta 1 fibronectin receptor during cell cycle withdrawal. Treatment of cultured cardiomyocytes with beta1 blocking antibody inhibited the cell cycle in fetal but not in neonatal cells. CONCLUSION: These results suggest that progression through the cardiomyocyte cell cycle may be dependent upon cell attachment via integrin beta1 and correlate with changes that occur in beta1 spliced variants and their respective alpha isoforms.

Animals↗
PubMed2003

Involvement of corneal nerves in the progression of keratoconus.

Keratoconus is a debilitating corneal thinning disease that principally develops in the second and third decades of life. Our group previously developed a novel approach to studying keratoconus, based on the observation that there is a gradient of damage across the keratoconic cone. We identified a number of cellular characteristics of keratoconus such as discrete incursions of fine cellular processes from the anterior keratocytes in association with localised indentation of the basal epithelium, and increased levels of the lysosomal enzymes Cathepsin B and G in aberrant keratocytes, located beneath compromised regions of Bowman's layer, but also deeper in the stroma. Enzyme activity by these cells seemed to be causing localised structural degradation of the anterior stroma, leading to near-complete destruction of both Bowman's layer and the stroma, often necessitating a full-thickness corneal graft for sight restoration. This current study extends our initial findings by investigating the role of corneal nerves passing between the stroma and epithelium at the sites of early degradative change observed previously, and may be facilitating the keratocyte-epithelial interactions in this disease. Cells in sections of normal and keratoconic human corneas were labelled with the fixable fluorescent viability dye 5-chloromethylfluorescein diacetate, antibodies to alpha-tubulin (nerves), alpha3beta1 integrin, Cathepsin B and G, and the nuclear dye DAPI, and then examined with a confocal microscope. Anterior keratocyte nuclei were seen wrapping around the nerves as they passed through the otherwise acellular Bowman's layer, and as the disease progressed and Bowman's layer degraded, these keratocytes were seen to express higher levels of Cathepsin B and G, and become displaced anteriorly into to the epithelium. Localised nerve thickenings also developed within the epithelium in association with Cathepsin B and G expression, and appeared to be very destructive to the cornea. Insight into the molecular mechanisms of keratoconic disease pathogenesis and progression can be gained from the process of extracellular matrix remodelling known from studies of connective tissues other than the cornea, and wound healing studies in the cornea. Further studies are required to determine how well this model fits the actual molecular basis of the pathogenesis of keratoconus.

Cathepsin B↗
PubMed1995

Expression of cadherins and integrins in human endometrium throughout the menstrual cycle.

OBJECTIVE: To detect cadherin and integrin expression in biopsies of endometrium in the phases of the cycle. Cell adhesion molecules may be involved in endometrial shedding during menstruation and attachment of shed endometrial tissue to the peritoneal lining in endometriosis patients. DESIGN: An immunohistochemical study on fresh frozen sections. SETTING: Tertiary-care university medical center. PATIENTS: Sixteen patients undergoing monitoring of their cycle as part of a subfertility workup. All patients had regular and ovulatory cycles. INTERVENTIONS: Endometrium samples were obtained at well-defined phases of the cycle. Simultaneously, blood samples were collected for E2 and P assay. MAIN OUTCOME MEASURES: The expression of cell adhesion molecules, including E- and P-cadherin and the integrins alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1, and the expression of estrogen receptor (ER) and P receptor (PR). RESULTS: E- and P-cadherin expression was demonstrated in all endometrium samples. Integrins alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1 were detected in samples from all cycle phases, whereas integrin alpha 2 beta 1 was not detected in midluteal samples. The serum levels of E2 were 24.7 pg/mL (range: 10.9 to 35.4 pg/mL) in the early follicular phase and 190.7 pg/mL (range: 152.5 to 256.1 pg/mL) in the preovulatory phase (conversion factor to SI unit, 3.671). Serum P was 13.7 ng/mL (range: 10.3 to 16.7 ng/mL) in the midluteal phase and 6.4 ng/mL (range: 1.2 to 13.7 ng/mL) in the premenstrual phase (conversion factor to SI unit, 3.180). The portion of cells staining for ER and PR was at a maximum during the preovulatory phase, both for epithelial and stromal cells. CONCLUSIONS: E- and P-cadherin expression was detected in all samples and did not vary throughout the menstrual cycle. If their expression is involved functionally in the cyclic menstrual shedding, the loss of expression is limited to a short period of time. Of the beta 1 integrins, only alpha 2 beta 1 expression was modulated during the menstrual cycle and found to be absent in the midluteal phase. No relation was found between the expression of cell adhesion molecules and the expression of ER and PR. Because the cadherins and beta 1 integrins could be detected in late luteal phase endometrium, these cell adhesion molecules could be involved in the attachment of endometrial fragments to the peritoneal lining as a result of retrograde menstruation. The potential function in the pathogenesis of endometriosis remains to be elucidated.

Antigens, CD↗
PubMedDate not supplied

The expression of laminin-5 and ultrastructure of the interface between basal cells and underlying stroma in the keratoconus cornea.

PURPOSE: We investigated the expression of laminin-5 and integrins, and the ultrastructure of the interface between basal cells and the basement membrane in the keratoconus cornea. These findings were compared to those in normal central cornea and limbus. METHODS: Frozen sections of the normal cornea (center and limbus) and the keratoconus cornea were immunostained with monoclonal antibodies against three chains of laminin-5 and integrins. To investigate the ultrastructure of the interface between basal cells and the underlying stroma, we used transmission electron microscopy. RESULTS: As compared to those in the normal central cornea, immunostaining patterns of the three chains of laminin-5 were thick and irregular in the keratoconus cornea and the normal limbus. Using electron microscopy analysis, the same characteristic structure of the interface between basal cells and the underlying stroma was recognized in the keratoconus cornea and the normal limbus. The expression of integrin alpha(6)beta(4) was restricted to the basal aspect of basal cells in the normal cornea. In the keratoconus cornea, however, integrin alpha(6)beta(4) was expressed in all aspects in basal and suprabasal cells. CONCLUSION The expression patterns of laminin-5 and the ultrastructure of the interface between basal cells and the basement membrane in the keratoconus cornea were similar to those in the normal limbus.

Adult↗
PubMed1997

Integrin regulation of polymorphonuclear leukocyte apoxis during hypoxia is primarily dependent on very late activation antigens 3 and 5.

BACKGROUND: Apoptosis is thought to be a central mechanism that leads to resolution of the inflammatory response. The regulation of polymorphonuclear leukocyte (PMN) apoptosis during hypoxia has not been previously characterized, and we hypothesized that integrin signaling by matrix proteins (laminin) would regulate PMN apoptosis. METHODS: PMNs at 1 x 10(5)/ml were adhered on plastic or laminin for 12 hours during normoxia or hypoxia. Apoptosis was determined both by cellular histologic evaluation and the TUNEL assays (Tdt). Phagocytosis in apoptotic PMNs was determined with two-color flow cytometric analyses with rhodamine-labeled heat-killed Escherichia coli (511 nm) and the Tdt reagent (563 nm). Western blot analyses were performed on nine apoptotic regulatory proteins with monoclonal antibodies directed against each protein, and tyrosine phosphorylation was assessed after integrin receptor cross-linkage. RESULTS: Adherence of PMNs to laminin reduced apoptosis by cellular histologic evaluation and the Tdt method (%apoptosis = 19 +/- 1.0 versus 63 +/- 4.2 by histologic evaluation, 38 +/- 3.8 versus 60 +/- 10.5 by flow cytometry +/- adherence to laminin). Apoptosis-positive PMNs exhibited significantly greater phagocytosis than apoptosis-negative PMNs +/- laminin. Western blot analyses demonstrated increased p53 expression after 2 and 4 hours of hypoxia. Cross-linkage of very late activation antigen-3 (alpha 3/beta 1) resulted in the phosphorylation of 53 kd, 44 kd, and 39 kd proteins at 30 seconds. CONCLUSIONS: (1) Chemotaxis of PMNs into the interstitium during hypoxia not only provides a means of ensuring PMN-pathogen contact but also provides a mechanism for improved survival by reducing apoptosis. (2) The reduction of apoptosis is mediated primarily by very late activation antigen-3, which leads to a subsequent increase in the intracellular expression of p53 and increased bacterial phagocytosis.

Apoptosis↗
PubMed2002

Molecular assembly of CD46 with CD9, alpha3-beta1 integrin and protein tyrosine phosphatase SHP-1 in human macrophages through differentiation by GM-CSF.

Human CD46, formerly membrane cofactor protein (MCP), binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic viral infection. CD46 on activated macrophages (Mphi) but not intact monocytes is presumed to be the factor responsible for virus-mediated immune modulation including down-regulation of IL-12 production. As CD46 is expressed on both Mphi and monocytes, the molecular mechanisms responsible for these distinct immune responses remain largely unknown. Here, we found that peripheral blood monocytes treated for 5--8 days with GM-CSF (i.e. mature Mphi) acquired the capacity to assemble CD9, alpha3-beta1 integrin and the tyrosine phosphatase SHP-1 with their CD46. Prior to this maturation stage, Mphi expressed sufficient amounts of CD9 and CD46 but showed no such complex formation, and as in intact monocytes MV replication was markedly suppressed. By flow cytometry and confocal microscopy, the complex was found to assemble on the surface in cells treated with approximately 6 days with GM-CSF but not for approximately 2 days. Notably, an alternative MV receptor SLAM CDw150 was neither expressed nor recruited to this complex throughout GM-CSF-mediated Mphi differentiation. These responses and molecular links were not reproduced in the hamster cell line CHO expressing human CD46 although these cells acquired high susceptibility to MV. Based on these observations, MV susceptibility in human myeloid lineages appears not to be as simple as that observed in human CD46-transfected non-myeloid cells. The molecular complex involving CD46 may confer high MV permissiveness leading to immune modulation in Mphi.

Animals↗
PubMed2000

Heparin-binding EGF-like growth factor: a juxtacrine growth factor.

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family growth factors, is synthesized as a membrane-anchored form (proHB-EGF). Proteolytic cleavage of proHB-EGF at the extracellular domain yields the soluble form of HB-EGF (sHB-EGF). ProHB-EGF is not only the precursor molecule for sHB-EGF but also a biologically active molecule itself. Recent studies indicate that proHB-EGF has unique properties distinct from the soluble form. ProHB-EGF forms a complex with membrane proteins including a tetramembrane spanning protein: CD9, an adhesion molecule integrin: alpha3beta1, and heparan sulfate proteoglycans. The complex is localized at the cell-cell contact site, suggesting that proHB-EGF may function in cell-to-cell signaling by a juxtacrine mechanism. In an in vitro model system, proHB-EGF showed growth inhibitory activity, while sHB-EGF was growth stimulatory. Ectodomain shedding, conversion of the membrane-anchored form into the soluble form, is regulated by multiple signaling pathways. All these characteristics imply that proHB-EGF and sHB-EGF are used in different ways. In vivo functions of sHB-EGF and proHB-EGF have been largely undefined, but recent studies implicate them in a variety of physiological processes including blastocyst implantation and wound healing.

Animals↗
PubMed2003

Mass spectrometric based mapping of the disulfide bonding patterns of integrin alpha chains.

Integrins are one of the major mediators of cellular adherence. Structurally the component alpha and beta chains are characterized by extensive intrachain disulfide bonding. The assignment of these bonds is currently based on homology with the chains of the integrin alphaIIbbeta3. However, recent crystallographic analysis of the soluble alphaVbeta3 construct indicates that the alphaV chain displays bonding patterns different from those predicted for alphaIIb. In an effort to define the disulfide bonding patterns in integrins, we have used mass spectrometric based approaches to map the human alpha3, alpha5, alphaV, and alphaIIb. The results indicate that there are differences in the disulfide patterns of the alpha chains. These do not correlate with the integrin capacity to bind ligands as all integrins used in the present study displayed functional activity. The differences were observed in the bonding patterns linking the heavy (H) and light (L) components of the of the alpha chains. It was also possible to assign the location in alpha5 of an additional disulfide bond involving a pair of cysteines not present in alphaV or alphaIIb. This second bond between the H and L chains of alpha5 has not been previously described. These results indicate that not all integrin species display the same disulfide bonding patterns. They also highlight the need for caution in the use of assignments based on sequence homology.

Amino Acid Sequence↗
PubMed1998

Recombinant soluble human alpha 3 beta 1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner.

Using insect cells, we expressed large quantities of soluble human integrin alpha 3 beta 1 ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble alpha 3 beta 1 specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble alpha 3 beta1 integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the alpha 3 beta 1 integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg2+, alpha 3 beta 1's binding affinity for invasin (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha 3 beta 1 ectodomain heterodimer resembles that of wild-type membrane-anchored beta 1 integrins. The integrin alpha 3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the alpha 3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant alpha 3 beta 1 integrin have provided new insights into alpha 3 beta1 structure, ligand binding function, specificity, and regulation.

Adhesins, Bacterial↗
PubMed1999

Alpha 3 beta 1 adhesion to laminin-5 and invasin: critical and differential role of integrin residues clustered at the boundary between alpha 3 N-terminal repeats 2 and 3.

Integrin/ligand interaction is a therapeutic target for many diseases. We previously reported that residues critical for ligand binding are clustered in N-terminal repeat 3 (in the predicted 2-3 loop) of alpha 4, alpha 5 and alpha IIb. Here we have localized residues critical for ligand binding in the alpha 3 subunit of integrin alpha 3 beta 1 with distinct ligand specificity (laminin-5). We identified an alpha 3 epitope common to several function-blocking anti-alpha 3 antibodies at the boundary between repeats 1 and 2 (residues 75-80). We found that swapping the predicted 4-1 loop (residues 153-165) at the boundary between repeats 2 and 3 with the corresponding alpha 4 sequence and mutating Thr-162 and Gly-163 residues in this predicted loop block laminin-5 binding. Thr-162 and Gly-163 and the antibody epitope are separated in the primary structure; however, they are close to each other in the proposed beta-propeller model. Mutating residues recently reported to block (Tyr-186 and Trp-188) or enhance (Asp-122) laminin-5 binding to alpha 3 beta 1 [Krukonis, E. S., Dersch, P., Eble, J. A., and Isberg, R. R.(1998) J. Biol. Chem. 273, 31837-31843] did not affect laminin-5 binding under the assay conditions used. Thr-162 and Gly-163 are not critical for adhesion to invasin, indicating that laminin-5 and invasin may use different recognition mechanisms, and that mutation of Thr-162 and Gly-163 does not drastically affect the integrity of alpha 3 beta 1. These results suggest that residues critical for ligand binding may be similarly (but not identically) located in repeat 3 of the alpha subunit regardless of ligand specificity.

Adhesins, Bacterial↗
PubMed1998

Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer.

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both alpha2beta1 and alpha3beta1 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequence intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhesion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients.

Adenocarcinoma, Scirrhous↗
PubMed1999

Ras-transfection up-regulated HaCaT cell migration: inhibition by Marimastat.

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-nis clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines.

Cell Adhesion↗
PubMed2000

A three-dimensional skin culture model for mouse keratinocytes: application to transgenic mouse keratinocytes.

The study of mouse epidermal biology has been hampered by the lack of a good in vitro model for the culture of mouse keratinocytes which allowed the reconstruction of a fully differentiated epidermis. We adapted the Pruniéras' model, also called the Dead de-Epidermized Dermis model (DED), to mouse keratinocytes and showed that a neo-epidermis can be reconstructed exhibiting a complete differentiation program. We also used this model to culture transgenic mouse keratinocytes. We observed that transgene expression occurred in the correct location and that the neo-epidermis mimed previous in vivo observations obtained with integrin skin-targeted transgenic mice. Therefore, this model will be a powerful tool to further investigate normal mouse and transgenic keratinocyte biology.

Animals↗