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PubMed2006

Ecto-5'-nucleotidase (eN, CD73) is coexpressed with metastasis promoting antigens in human melanoma cells.

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: alpha5-, beta1-, beta3-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. alphav-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.

5'-Nucleotidase↗

PubMed2005

Integrin alpha3beta1 suppresses long-term potentiation at inhibitory synapses on the cerebellar Purkinje neuron.

At the GABAergic synapses between inhibitory interneurons and a Purkinje neuron (PN) in the cerebellum, the postsynaptic depolarization induces the long-term potentiation (called rebound potentiation; RP) of GABAA receptor responsiveness. Here, we show that integrins, a type of cell-adhesion molecules, are involved in the regulation of RP. Integrin activation by Mn2+ impaired the RP induction of GABA responsiveness and mIPSCs in PNs, which was abolished by the function blocking antibody against either integrin alpha3 or beta1 subunit, but not by that against alpha5 or alphaV subunit. Furthermore, overexpression of integrin alpha3 subunit in a PN by itself impaired the RP induction. We also show that Src-family of protein tyrosine kinases mediated the suppressive effect of integrin activity on the RP induction. Thus, the integrin/Src pathway negatively regulates the induction of long-term plasticity at inhibitory synapses on a cerebellar PN.

Animals↗

PubMed1998

Epidermal integrin expression is upregulated rapidly in human fetal wound repair.

BACKGROUND/PURPOSE: The fetus heals skin wounds rapidly and scarlessly. The mechanisms that mediate the rapid reepithelialization that is seen in this process are unknown. Integrins are a family of cell surface receptors that bind fibronectin, tenascin, collagen, and other extracellular matrix proteins that are deposited rapidly in fetal wounds. The authors hypothesized that epidermal integrin receptors specific for fibronectin and other wound matrix proteins are upregulated rapidly during human fetal repair. METHODS: To investigate the spatial and temporal expression of integrins in scarless fetal repair, fetal skin from six human abortuses (16 to 23 weeks' gestation) was transplanted subcutaneously into severe combined immunodeficient mice. After graft take, full-thickness incisional wounds were made in the grafts, and grafts were harvested at various time-points from 4 hours to 28 days after wounding. Integrin receptor protein expression was analyzed at each time-point using immunohistochemistry with monoclonal antibodies specific for the receptors that bind fibronectin, tenascin, collagen, and laminin (alpha5, alpha(v), beta6, alpha2, alpha3, alpha6, and beta4). RESULTS: In this model, wounded human fetal skin grafts reepithelialized rapidly (within 24 to 36 hours) and healed scarlessly. Within 4 hours of wounding, the grafts showed increased, suprabasal expression (alpha2, alpha3, alpha6, beta4) or neoexpression (alpha5, alpha(b), beta6) of integrins at the epidermal wound edge. This increased expression persisted until reepithelialization was complete. CONCLUSIONS: Early upregulation of integrins in fetal wounds may permit rapid keratinocyte migration and reepithelialization, and may be important in limiting the induction of inflammatory mediators and scar.

Animals↗

PubMed1999

R-Ras signals through specific integrin alpha cytoplasmic domains to promote migration and invasion of breast epithelial cells.

Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the alpha2, but not the alpha5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin beta cytoplasmic domains was observed for the alpha2 cytoplasmic domain; alpha2beta1-mediated migration was inhibited by the expression of excess alpha2 but not alpha5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin alpha subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin alpha cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion.

Breast↗

PubMed2004

Epidermal growth factor differentially affects integrin-mediated adhesion and proliferation of ACL and MCL fibroblasts.

The anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) are two commonly injured structures in the human knee. While the MCL heals post-traumatically, the ACL does not. Since growth factors play a major role in the proliferation phase of wound healing, we compared the differential effects of epidermal growth factor (EGF) on adhesion and proliferation of ACL and MCL fibroblasts. Using a micropipette/micromanipulator system we found that cells subjected to shorter incubation periods (15 minutes) with EGF (5, 10, 50 ng/ml) showed a general increase in adhesion to the extracellular matrix fibronectin while cells subjected to longer incubation periods (4, 6, 10, and 24 hr) with EGF (5 ng/ml) showed decreases in adhesion. For both incubation durations, MCL fibroblasts displayed a greater change in adhesion than ACL fibroblasts, when compared to control. Investigation of integrin expression showed no fluctuation in cell surface expression of the alpha5 subunit of the FN-binding integrin alpha5beta1 for all EGF (5 ng/ml) incubation times. MCL cells showed a significant increase in proliferation upon stimulation with EGF compared to ACL cells when cultured in FN coated wells. The results found in this study help provide a better understanding of EGF's role in adhesion and proliferation, two events that may contribute to the differential healing response between ACL and MCL fibroblasts. Following exposure to EGF, ACL and MCL cells initially respond by increasing their adhesion strength. MCL cells respond to all concentrations of EGF while ACL cells appear to have a threshold concentration after which EGF effects plataeu. After this initial response period (<10 hr) cells exhibit lower adhesion strength and higher proliferation rate. It is possible that the release from FN serves to enhance the ability of the cells to proliferate. These results may aid in understanding the ligament healing process.

Adult↗

PubMed2004

Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins.

Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the alpha5, alphaV, beta1, and beta3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells.

Adhesins, Bacterial↗

PubMed1998

Integrins in thyroid tissue: upregulation of alpha2beta1 in anaplastic thyroid carcinoma.

OBJECTIVE: To evaluate the integrin pattern in the normal thyroid gland and in different pathological disorders including malignant tumors, because the aggressiveness of several malignant tumors correlates with alterations in the expression of one or more integrins. DESIGN: We examined the expression of integrins and E-cadherin immunohistochemically in a large and well-defined sample of normal and pathological human thyroid tissue. METHODS: Cryosections of 58 thyroid tissue specimens from normal tissue, thyrotoxicosis, nodular goiter, oxyphilic adenoma, follicular adenoma, follicular carcinoma, papillary carcinoma and anaplastic carcinoma, and three lymph node metastases were investigated immunohistochemically using monoclonal antibodies specifically recognizing the integrin beta1-, beta4-, alpha1-, alpha2-, alpha3-, alpha5- and alpha6-subunits, or E-cadherin. RESULTS: All thyroid epithelial cells expressed integrin beta1- and alpha3-subunits. Immunostaining of the beta4-subunit and the alpha6-subunits was found only in tumors. The staining pattern in the three lymph node metastases from papillary carcinomas did not differ from that in their primaries. Anaplastic carcinomas demonstrated neoexpression of the integrin alpha2-subunit. E-cadherin was detected in all tissues except anaplastic carcinomas. CONCLUSIONS: Neoexpression of alpha6beta4 was seen in most malignant tumors, whereas alpha2 was exclusively found in anaplastic carcinomas. In the latter, a loss of E-cadherin expression was also seen. These changes in cell adhesion molecule expression strongly suggest an association with the acquisition of proliferative and invasive properties.

Cadherins↗

PubMed2003

Defective integrin switch and matrix composition at alpha 7-deficient myotendinous junctions precede the onset of muscular dystrophy in mice.

Force transmission at the myotendinous junction requires a strong link between the muscle cytoskeleton and the extracellular matrix. At the adult junction, two splice variants of the laminin-binding integrins, alpha7Abeta1D and alpha7Bbeta1D, are highly enriched. The alpha7 subunits are critical for the integrity of the junctional sarcolemma because integrin alpha7-deficient mice develop muscular dystrophy, primarily affecting this site of the muscle. Here, we report that beta1D integrin coimmunoprecipitates and colocalizes with the alpha5 subunit at alpha7-deficient junctions, but does not associate with alpha3, alpha6 or alphav integrins. By immunogold labelling we show that the basement membranes of integrin alpha7-deficient muscles recruit abnormally high levels of fibronectin, the ligand of alpha5beta1D. Finally, we demonstrate that alpha5beta1D is down-regulated at the normal postnatal junction and is displaced by alpha7beta1D. These results suggest that the alpha7 subunit is implicated in the down-regulation of alpha5beta1D and in the removal of fibronectin from the maturing myotendinous junction, thus providing an alpha7beta1D-based link to laminin. We propose that the persistence of alpha5beta1D in alpha7-deficient mice is not compatible with normal muscle function and leads to muscle wasting.

Animals↗

PubMed1999

Expression of beta1 and beta4 integrins in normal arachnoid membrane and meningiomas.

BACKGROUND: The aim of this work was to study the expression of alpha, beta1, and beta4 integrin subunits in meningiomas. METHODS: Seventeen atypical or anaplastic meningiomas were retrieved from the files of Hôpital de Bellevue, Saint-Etienne, France. They were compared with 17 benign meningiomas consecutively examined in 1997 and 6 schwannomas. The tumors were classified according to standard histologic criteria. Frozen sections were immunostained for alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, beta1, and beta4 integrin subunits; collagen; laminin and fibronectin; cytokeratin; vimentin; neural cell adhesion molecule (NCAM); and MIB-1. RESULTS: The study included 7 fibrous meningiomas, 6 transitional meningiomas, 19 syncytial meningiomas, and 2 secretory meningiomas. The expression of alpha1, alpha3, alpha5, alpha6, and beta1 was constant. The expression of alpha1 was higher in fibrous meningiomas than in syncytial meningiomas. Only in transitional, syncytial, and secretory meningiomas was the expression of alpha2 detected. The expression of alpha2 and beta4 was associated with the expression of cytokeratin in the glandular structures of secretory meningiomas, whereas it was associated with NCAM expression in the whorls of meningothelial meningiomas. The expression of integrin receptors by tumor cells was strongly correlated with that of their respective ligands in the extracellular matrix. In invasive meningiomas, the expression of alpha3 and alpha6 by tumor cells was significantly lower. The higher the MIB-1 proliferation index, the lower the expression of alpha3. The 6 schwannomas expressed only alpha2, alpha3, alpha6, beta1, and beta4 integrins. CONCLUSIONS: Each histologic subtype of meningioma has a specific spectrum of integrin expression. The study of alpha3 and alpha6 may have prognostic value in the assessment of meningiomas. The study of the integrin profile is valuable for the differential diagnosis of fibrous meningiomas and schwannomas.

Antigens, Nuclear↗

PubMed1999

A distinct integrin-mediated phagocytic pathway for extracellular matrix remodeling by RPE cells.

PURPOSE: To characterize the phagocytosis of extracellular matrix components by retinal pigment epithelial cells and to determine which receptors and signal transduction pathways are involved. METHODS: Fluorescent latex beads were coated with fibronectin (FN), collagen type I or IV, or thrombospondin and incubated with human retinal pigment epithelial cells for 3 hours. Phagocytosis was quantified by flow cytometry. The effects of adhesion blocking antibodies to cell surface receptors (alpha1, alpha3, alpha5, beta1, alpha5beta1, alphavbeta3, alphavbeta5 integrins and CD36) and inhibitors of specific intracellular signaling pathways (tyrosine kinase phosphatidylinositol 3-kinase [PI3-kinase], protein kinase C [PKC], and mitogen-activated protein kinase) were determined using FN-coated beads. RESULTS: Phagocytosis of FN-coated beads was greater than phagocytosis of beads coated with collagen type I, collagen type IV, or thrombospondin or uncoated controls (P < 0.0005). Anti-alpha5, -beta1, and -alpha5beta1 antibodies markedly inhibited FN phagocytosis (P < 0.0005); the inhibitory effects of anti-alpha5 antibody were stronger in the initial stages (binding) than in the later stages (internalization) of phagocytosis. There was no significant effect on phagocytosis when anti-alpha1, -alpha3, -alphavbeta5, -alphavbeta3 or -CD36 antibodies were used. Fibronectin phagocytosis was decreased by inhibitors of tyrosine kinase (genistein, 100 microg/ml, P < 0.005) and PI3-kinase (wortmannin, 5 microM, P < 0.01), but these reagents did not affect the uncoated controls. The PKC inhibitor calphostin C (400 nM) nonspecifically increased the phagocytosis of FN-coated (P < 0.05) and uncoated beads (P < 0.01). CONCLUSIONS: Subconfluent retinal pigment epithelial cells preferentially phagocytose FN over other extracellular matrix components. Phagocytosis of FN utilizes the alpha5beta1 integrin, is mediated in part through tyrosine kinase and PI3-kinase signaling pathways, and is modulated by PKC. Phagocytosis of extracellular matrix by retinal pigment epithelial cells may represent a novel mechanism for remodeling of the provisional extracellular matrix during outer retinal wound healing.

CD36 Antigens↗

PubMed1998

Amniotic membrane as an in vitro model for endometrium-extracellular matrix interactions.

The aim of the study was to develop an in vitro model to study the interaction between endometrial cells and extracellular matrix (ECM) and to evaluate the expression of cell adhesion molecules in endometrial cells and tissue fragments under in vitro conditions. Endometrial biopsies were collected from 32 patients. Samples were either digested using collagenase type I, or dissected mechanically. Adhesion of isolated cells and tissue fragments to stripped amniotic membranes and to coverslips coated with ECM components was studied. The expression of beta1 integrins and cadherins was assessed using immunohistochemistry. Collagenase digestion of endometrial biopsies yielded viable single cells. These cells did not adhere to either side of stripped amniotic membranes, and did not show expression of the cell adhesion molecules. In contrast, mechanically fragmented endometrium samples adhered to both sides of stripped amniotic membranes and showed immunohistochemical expression of E- and P-cadherin and integrin subunits alpha2, alpha3, alpha4, alpha5 and alpha6. Amniotic membranes, after stripping of epithelial lining, are suitable to study interactions between endometrial tissue and ECM in functional and structural studies. Endometrial cells after collagenase type I digestion do not adhere to stripped amniotic membrane and have lost expression of beta1 integrins and E- and P-cadherin.

Amnion↗

PubMed2003

Expression and regulation of integrin receptors in human trophoblast cells: role of estradiol and cytokines.

Embryo implantation and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-cell communication amongst the implanting blastocyst and the receptive endometrium through integrins, a large family of proteins involved in the attachment, migration, invasion and control of cellular functions. Integrins display dynamic temporal and spatial patterns of expression by the trophoblast cells during early pregnancy in humans. However, the precise mechanism of embryo implantation and the modulation of the integrin receptors during blastocyst attachment and further implantation remain elusive in the humans. The present study elucidates the expression and hormonal modulation of fibronectin, vitronectin and laminin integrin receptors by estradiol and IL-1alpha in human trophoblast cells. Human first trimester trophoblast cells showed the induction of the classical estrogen receptor (ER)-alpha by its own ligand, estradiol. Treatment with either estradiol or IL-1alpha induced the expressions of alpha4, alpha5, alpha6 and alpha(v) integrin receptor subunits at both the mRNA and protein levels, while expression of beta1 remained unaltered. Furthermore, estradiol upregulated the expression of IL-1alpha, thereby suggesting the possibility that estrogen may either directly or via the proinflammatory cytokine induces the expression of the cell surface integrin receptors. The findings delineate the role of hormones and the cytokines in modulating the adhesiveness and attachment of the trophoblast cells. This may reflect the in vivo scenario where the implanting embryo is surrounded by a hormone-cytokine rich uterine microenvironment that may precisely regulate the expression of integrins and thereby facilitate implantation.

Cytokines↗

PubMed1999

Modulation of integrin expression on mesotheliomas: the role of different histotypes in invasiveness.

The in vitro and in vivo integrin expression in human pleural malignant mesothelioma (MM) of three different histotypes was studied. Cell lines from MM of epithelioid (E1), fibrous (F1), byphasic histotype (B1) and normal mesothelial cells (NM) were analysed for the surface expression of alpha2, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, beta4 subunits and alphavbeta5 integrins. We found that alpha6, beta4 subunits and alphavbeta5, weakly detectable on NM cells, were expressed on MM cells. The beta3 subunit, well expressed on NM cells, was absent on MM cells. Differential expression among histotypes was observed, the MM-E1 was the least and the MM-B1 the most positive. Specimens for each MM histotype, were analysed by immunohistochemistry. The alpha6 and alphav subunits were more evident on the epithelioid histotype. Intense staining for beta3 and beta4 subunits, was found in all MM, particularly in invading cells, while the alpha5, and alphavbeta5 integrins were variously expressed. The different histotypes can affect the in vitro integrin expression and may indicate a preferential involvement of some subunits in vivo during MM tumor progression.

Humans↗

PubMed2006

Tyrosine kinase inhibitors alter adhesivity of prostatic cancer cells to extracellular matrix components.

Tyrosine kinase inhibitors (TKIs) are thought to have potential as a new generation of anti-cancer drugs. Since invasiveness, the main characteristic of malignant behaviour, is believed to depend on altered cell-matrix interactions, we investigated the effect of two potent TKIs, genistein and tyrphostin AG-1478, on the interaction of prostate cancer cells with extracellular matrix components. PC-3 and DU-145 cells were treated with various concentrations of genistein and tyrphostin AG-1478. Adhesion to extracellular matrix was assayed using fluorescence-labelled cells seeded on collagen type I, collagen type IV, fibronectin, laminin and vitronectin. The expression levels of integrin beta1, alpha2, alpha3 and alpha5 subunits were measured using flow cytometry of cells labelled with monoclonal murine antibodies. Genistein treatment reduced the ability of both cell lines to adhere to the matrix proteins tested. This effect was more pronounced for PC-3 cells than for DU-145 cells. Genistein treatment decreased the expression of beta1 integrins by 40% in PC-3 cells and 22% in DU-145. AG-1478 treatment slightly reduced the ability of DU-145 cells to adhere, but did not decrease PC-3 cell adhesion. Nevertheless, expression levels were reduced for most integrins tested, except the expression of alpha-5, for which no significant effect was measured. Our results point to a possible role of TKIs as suppressors of prostate carcinoma cell adhesion to extracellular matrix components, by acting as inhibitors of integrin expression.

Cell Adhesion↗

PubMed2006

Mixture of sugar and povidone--iodine stimulates wound healing by activating keratinocytes and fibroblast functions.

The topical application of a mixture of sugar and povidone--iodine (PI) has been reported to accelerate the healing of cutaneous wounds and ulcers by promoting re-epithelialization and granulation tissue formation as well as having an anti-microbial effect. To clarify the mechanisms accounting for the efficacy of a 70% sugar and 3% PI paste (U-PASTAtrade mark) (SP), various keratinocytes and fibroblasts functions, including proliferation, collagen synthesis, integrin expression, and cytokine and proteinase secretions in the presence of SP were investigated. Cultured human keratinocytes and fibroblasts were treated with various concentrations of SP, SU and PI. The secretion of urokinase-type plasminogen activator (u-PA), transforming growth factor (TGF)-alpha and interleukin-1alpha from keratinocytes, was detected by ELISA. Collagen synthesis of fibroblasts was examined by means of detecting proline uptake. Furthermore, integrin expressions of these cells were analyzed using a flow cytometer. SP and PI increased intra-cellular u-PA of keratinocytes and stimulated the secretion of u-PA and TGF-alpha. Sugar accelerated the extra-cellular u-PA level only. Both SP and sugar increased the collagen synthesis of fibroblasts. SP and PI also remarkably induced the expressions of extra-cellular matrix receptor integrins, alpha1, alpha2, alpha3, alpha4, alpha5 and beta1, on the surface of keratinocytes and fibroblasts. SP, the mixture of sugar and PI, is likely to act on wounds not only as an antibiotic agent, but also as a modulator for keratinocytes and fibroblasts.

Anti-Infective Agents↗

PubMed1998

Activation of the sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation.

The proliferative response of hematopoietic cells is regulated by many factors, including the presence and type of growth factors, the cellular microenvironment, and the physiochemical conditions prevailing in the tissue milieu. A process fundamental to all cells is the regulation of the intracellular acid-base conditions. One of the mechanisms by which intracellular pH (pHi) is regulated is through the sodium/hydrogen exchanger, a ubiquitous membrane protein which exploits the intra- and extracellular sodium ion gradient to drive hydrogen ions out of the cell. However, activation of the exchanger via mitogenic and nonmitogenic signals leads to an increase in pHi which, in turn, may directly or indirectly result in a proliferative response. It has been shown that interaction of fibronectin with its integrin receptor subunits alpha4 and alpha5 can result in activation of the Na+/H+ exchanger. In this report, we demonstrate that when mouse bone marrow cells are physically brought together in a preculture system we designate as high cell density culture (HCDC), in a small volume and at the same cellularity as that in the marrow, hematopoietic stem and progenitor cell populations are stimulated with no additional stimulation in the presence of growth factors. Neutralizing antibodies to the growth factors added to HCDC had little, if any, effect on the degree of stimulation. However, when antibodies to fibronectin or the alpha4 integrin subunit were added to HCDC, inhibition was observed, indicating that the observed hematopoietic stimulation occurred via the fibronectin-integrin pathway. Addition of 5 microM 5-(N,N-hexamethylene) amiloride (5-HMA), a specific inhibitor of the Na+/H+ exchanger, also resulted in inhibition of in vitro hematopoiesis. Since the exchanger was implicated, we then measured the pHi of normal and HCDC-treated bone marrow cells in the absence and presence of 5-HMA by flow cytometry using the fluorescent pH-sensitive indicator, carboxy SNARF-1 AM. It was found that cells subjected to HCDC exhibited a higher pH than normal fresh cells. In each case, the pH was lowered in the presence of 5-HMA. Furthermore, addition of antibodies to fibronectin or the alpha4 integrin subunit to HCDC also reduced the pH, to a similar level to that found for 5-HMA. Our results demonstrate, for the first time, that a hematopoietic stem and progenitor cell proliferative response can be initiated by activation of the Na+/H+ exchanger, leading to an increase in pHi, via cell-cell interaction through the fibronectin-integrin pathway. This pathway could, therefore, be significant not only in normal hematopoietic regulation, but also under pathophysiological conditions.

Amiloride↗

PubMed2005

Probing the integrin-binding site within the globular domain of laminin-511 with the function-blocking monoclonal antibody 4C7.

In an attempt to elucidate the integrin-binding site within laminin-511 (alpha5beta1gamma1), we mapped the epitope for mAb 4C7, which recognizes the globular (G) domain of the laminin alpha5 chain and inhibits binding of integrin alpha6beta1 to laminin-511, using a series of recombinant laminin-511 mutants with deletions or substitutions in the G domain. Deletion of the LG2-5 modules only partially compromised the 4C7 binding activity, while deletion of all 5 LG modules completely abrogated the activity, indicating that the epitope for 4C7 resides in the LG1 module. In support of this conclusion, 4C7 reactivity was abolished when the LG1 module of laminin-511 was swapped with the corresponding module of laminin-111, but the reactivity was retained after swapping the LG2 or LG3 module. Despite the requirement of LG1 for 4C7 binding, a recombinant LG1 module failed to bind to 4C7 when expressed alone or in tandem with LG2, but exhibited significant 4C7 binding activity when expressed as an array of LG1-3. These results indicate that 4C7 recognizes an epitope in the LG1 module, whose active conformation is stabilized in the context of the LG1-3 modules. Despite their 4C7 binding activities, neither the recombinant LG1-3 fragment nor the LG2 and LG3 swap mutants were capable of binding to integrin alpha6beta1. Thus, the integrin binding activity does not necessarily parallel the 4C7 reactivity, and possibly requires a strictly defined conformation of the LG1 module which can only be attained within an array of the intact LG1-3 modules connected to the preceding coiled-coil domain.

Amino Acid Sequence↗

PubMed2003

Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture.

The authors have previously documented decreased epithelial basement membrane (BM) components and alpha3beta1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar-collagen gel at the air-liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 alpha5 chain, nidogen-1/entactin, integrin alpha3beta1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of alpha3beta1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized alpha3beta1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.

Aged↗