Improving Osteoblast Response In Vitro by a Nanostructured Thin Film with Titanium Carbide and Titanium Oxides Clustered around Graphitic Carbon.

INTRODUCTION: Recently, we introduced a new deposition method, based on Ion Plating Plasma Assisted technology, to coat titanium implants with a thin but hard nanostructured layer composed of titanium carbide and titanium oxides, clustered around graphitic carbon. The nanostructured layer has a double effect: protects the bulk titanium against the harsh conditions of biological tissues and in the same time has a stimulating action on osteoblasts. RESULTS: The aim of this work is to describe the biological effects of this layer on osteoblasts cultured in vitro. We demonstrate that the nanostructured layer causes an overexpression of many early genes correlated to proteins involved in bone turnover and an increase in the number of surface receptors for α3ß1 integrin, talin, paxillin. Analyses at single-cell level, by scanning electron microscopy, atomic force microscopy, and single cell force spectroscopy, show how the proliferation, adhesion and spreading of cells cultured on coated titanium samples are higher than on uncoated titanium ones. Finally, the chemistry of the layer induces a better formation of blood clots and a higher number of adhered platelets, compared to the uncoated cases, and these are useful features to improve the speed of implant osseointegration. CONCLUSION: In summary, the nanostructured TiC film, due to its physical and chemical properties, can be used to protect the implants and to improve their acceptance by the bone.

Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor ß Early Protransforming Events in Breast Epithelial Cells.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor ß (TGF-ß) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin ß1 mRNA, which drives the translationally controlled assembly of a TGF-ß receptor signaling complex containing α3ß1 integrins, ß-catenin, TGF-ß receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-ß. TGF-ß can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-ß, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-ß. Overexpression of eIF4E may be a proinvasive facilitator of TGF-ß activity.

Notoginsenoside R1 ameliorates podocyte adhesion under diabetic condition through α3ß1 integrin upregulation in vitro and in vivo.

BACKGROUND: Decreased expression of α3ß1 integrin may contribute to reduction in podocyte adhesion to glomerular basement membrane (GBM), which represents a novel early mechanism leading to diabetic kidney disease (DKD). Here, we examined the protective effects of Notoginsenoside R1 (NR1) on podocyte adhesion and α3ß1 integrin expression under diabetic condition in vitro and in vivo. METHODS: Conditionally immortalized mouse podocytes were exposed to high glucose (HG) with 10 and 100µg /ml of NR1 for 24 h. Podocyte adhesion, albuminuria, oxidative markers, renal histopathology, podocyte number per glomerular volume, integrin-linked kinase (ILK) activity and α3ß1 integrin expression were measured in vitro and in vivo. RESULTS: HG decreased podocyte adhesive capacity and α3ß1 integrin expression, the main podocyte anchoring dimer to the GBM. However, NR1 ameliorated impaired podocyte adhesive capacity and partially restored α3ß1 integrin protein and mRNA expression. These in vitro observations were confirmed in vivo. In streptozotocin(STZ)-induced diabetic rats, treatment with NR1 (5 and 10 mg· kg(-1)· d(-1)) for 12 weeks partially restored the number of podocytes per glomerular volume and glomerular α3ß1 integrin expression, as well as ameliorated albuminuria, histopathology and oxidative stress. NR1 also inhibited glomerular ILK activity in diabetic rats. CONCLUSION: NR1, a novel antioxidant, ameliorated glucose-induced impaired podocyte adhesive capacity and subsequent podocyte depopulation partly through α3ß1 integrin upregulation. These findings might provide a potential new therapeutic option for the treatment of DKD.

Angiopoietin 2 induces pericyte apoptosis via α3ß1 integrin signaling in diabetic retinopathy.

Pericyte loss is an early characteristic change in diabetic retinopathy (DR). Despite accumulating evidence that hyperglycemia-induced angiopoietin 2 (Ang2) has a central role in pericyte loss, the precise molecular mechanism has not been elucidated. This study investigated the role of Ang2 in pericyte loss in DR. We demonstrated that pericyte loss occurred with Ang2 increase in the diabetic mouse retina and that the source of Ang2 could be the endothelial cell. Ang2 induced pericyte apoptosis via the p53 pathway under high glucose, whereas Ang2 alone did not induce apoptosis. Integrin, not Tie-2 receptor, was involved for Ang2-induced pericyte apoptosis under high glucose as an Ang2 receptor. High glucose changed the integrin expression pattern, which increased integrin α3 and ß1 in the pericyte. Furthermore, Ang2-induced pericyte apoptosis in vitro was effectively attenuated via p53 suppression by blocking integrin α3 and ß1. Although intravitreal injection of Ang2 induced pericyte loss in C57BL/6J mice retina in vivo, intravitreal injection of anti-integrin α3 and ß1 antibodies attenuated Ang2-induced pericyte loss. Taken together, Ang2 induced pericyte apoptosis under high glucose via α3ß1 integrin. Glycemic control or blocking Ang2/integrin signaling could be a potential therapeutic target to prevent pericyte loss in early DR.

Dendritic cells: In vitro culture in two- and three-dimensional collagen systems and expression of collagen receptors in tumors and atherosclerotic microenvironments.

Dendritic cells (DCs) are immune cells found in the peripheral tissues where they sample the organism for infections or malignancies. There they take up antigens and migrate towards immunological organs to contact and activate T lymphocytes that specifically recognize the antigen presented by these antigen presenting cells. In the steady state there are several types of resident DCs present in various different organs. For example, in the mouse, splenic DC populations characterized by the co-expression of CD11c and CD8 surface markers are specialized in cross-presentation to CD8 T cells, while CD11c/SIRP-1α DCs seem to be dedicated to activating CD4 T cells. On the other hand, DCs have also been associated with the development of various diseases such as cancer, atherosclerosis, or inflammatory conditions. In such disease, DCs can participate by inducing angiogenesis or immunosuppression (tumors), promoting autoimmune responses, or exacerbating inflammation (atherosclerosis). This change in DC biology can be prompted by signals in the microenvironment. We have previously shown that the interaction of DCs with various extracellular matrix components modifies the immune properties and angiogenic potential of these cells. Building on those studies, herewith we analyzed the angiogenic profile of murine myeloid DCs upon interaction with 2D and 3D type-I collagen environments. As determined by PCR array technology and quantitative PCR analysis we observed that interaction with these collagen environments induced the expression of particular angiogenic molecules. In addition, DCs cultured on collagen environments specifically upregulated the expression of CXCL-1 and -2 chemokines. We were also able to establish DC cultures on type-IV collagen environments, a collagen type expressed in pathological conditions such as atherosclerosis. When we examined DC populations in atherosclerotic veins of Apolipoprotein E deficient mice we observed that they expressed adhesion molecules capable of interacting with collagen. Finally, to further investigate the interaction of DCs with collagen in other pathological conditions, we determined that both murine ovarian and breast cancer cells express several collagen molecules that can contribute to shape their particular tumor microenvironment. Consistently, tumor-associated DCs were shown to express adhesion molecules capable of interacting with collagen molecules as determined by flow cytometry analysis. Of particular relevance, tumor-associated DCs expressed high levels of CD305/LAIR-1, an immunosuppressive receptor. This suggests that signaling through this molecule upon interaction with collagen produced by tumor cells might help define the poorly immunogenic status of these cells in the tumor microenvironment. Overall, these studies demonstrate that through interaction with collagen proteins, DCs can be capable of modifying the microenvironments of inflammatory disease such as cancer or atherosclerosis.

Integrin α3ß1 can function to promote spontaneous metastasis and lung colonization of invasive breast carcinoma.

UNLABELLED: Significant evidence implicates α3ß1 integrin in promoting breast cancer tumorigenesis and metastasis-associated cell behaviors in vitro and in vivo. However, the extent to which α3ß1 is actually required for breast cancer metastasis remains to be determined. We used RNA interference to silence α3 integrin expression by approximately 70% in 4T1 murine mammary carcinoma cells, a model of aggressive, metastatic breast cancer. Loss of α3 integrin reduced adhesion, spreading, and proliferation on laminin isoforms, and modestly reduced the growth of orthotopically implanted cells. However, spontaneous metastasis to lung was strikingly curtailed. Experimental lung colonization after tail vein injection revealed a similar loss of metastatic capacity for the α3-silenced (α3si) cells, suggesting that critical, α3-dependent events at the metastatic site could account for much of α3ß1's contribution to metastasis in this model. Reexpressing α3 in the α3si cells reversed the loss of metastatic capacity, and silencing another target, the small GTPase RhoC, had no effect, supporting the specificity of the effect of silencing α3. Parental, α3si, and α3-rescued cells, all secreted abundant laminin α5 (LAMA5), an α3ß1 integrin ligand, suggesting that loss of α3 integrin might disrupt an autocrine loop that could function to sustain metastatic growth. Analysis of human breast cancer cases revealed reduced survival in cases where α3 integrin and LAMA5 are both overexpressed. IMPLICATIONS: α3 integrin or downstream effectors may be potential therapeutic targets in disseminated breast cancers, especially when laminin α5 or other α3 integrin ligands are also over-expressed.

The C-terminal region of NELL1 mediates osteoblastic cell adhesion through integrin α3ß1.

NELL1 is a secretory osteogenic protein containing several structural motifs that suggest that it functions as an extracellular matrix component. To determine the mechanisms underlying NELL1-induced osteoblast differentiation, we examined the cell-adhesive activity of NELL1 using a series of recombinant NELL1 proteins. We demonstrated that NELL1 promoted osteoblastic cell adhesion through at least three cell-binding domains located in the C-terminal region of NELL1. Adhesion of cells to NELL1 was strongly inhibited by function-blocking antibodies against integrin α3 and ß1 subunits, suggesting that osteoblastic cells adhered to NELL1 through integrin α3ß1. Further, focal adhesion kinase activation is involved in NELL1 signaling.

ADAM15 to α5ß1 integrin switch in colon carcinoma cells: a late event in cancer progression associated with tumor dedifferentiation and poor prognosis.

ADAM15, a member of the A Disintegrin And Metalloproteinase (ADAM) family, is a membrane protein containing an adhesion domain that binds to α5ß1 integrin through a unique RGD domain. ADAM15, expressed by human normal colonocytes, is involved in epithelial wound healing and tissue remodeling in inflammatory bowel disease. The aims of our study were (i) to analyze ADAM15 expression in a series of colon carcinomas and paired normal mucosa and (ii) to integrate the spatial relationship of ADAM15 with its binding partners α5ß1 integrin, a mesenchymal marker, as well as with other adhesion molecules, α3ß1 integrin and E-cadherin. A series of 94 colon carcinomas of the non other specified category were graded according to the World Health Organization classification. Immunohistochemistry was performed on frozen tissue sections using antibodies directed to ADAM15, α5ß1 and α3ß1 integrins, and E-cadherin. ADAM15 was quantified at the mRNA level. Finally, promoter methylation of ADAM15 was examined as well as the microsatellite instability status (MSS/MSI). Thirty-six percent of colorectal carcinomas displayed a reduced expression of ADAM15 in cancer cells, confirmed at the mRNA level in most cases, without promoter methylation. ADAM15 down-regulation was associated with histologically poorly differentiated carcinomas. In addition, it was associated with the acquisition of α5ß1 by cancer cells and down-regulation of α3ß1 integrin and E-cadherin. Finally this profile that includes characteristic of epithelial to mesenchymal transition is a late progression event of colon cancer with a poor prognosis.

Spironolactone ameliorates podocytic adhesive capacity via restoring integrin alpha 3 expression in streptozotocin-induced diabetic rats.

Podocyte responses to various injuries include detachment from the glomerular basement membrane (GBM) with impaired adhesion ability. Growing evidence suggests inappropriately enhanced aldosterone levels in glomeruli may contribute to podocytic injury and subsequently glomerulosclerosis in diabetic nephropathy (DN). In the present study, we aimed to investigate podocytic integrin alpha 3 expression and urinary podocyte excretion in streptozotocin (STZ)-induced diabetic rats, and to evaluate their responses to spironolactone (SPL). STZ-induced male diabetic Wistar rats were treated with vehicle (the STZ group, n=7), or spironolactone (the STZ+SPL group, n=6) for 12 weeks, six additional rats of similar body weight serving as control. Urine specimens were obtained for measurement of urine albumin concentration and urinary podocyte quantitation upon completion of the 12 weeks. Urinary podocyte excretion was quantified by immunofluorescence and expression of integrin alpha 3 was detected by immunohistochemistry and Western blotting. At 12 weeks, rats given STZ alone revealed an increase in blood glucose and were unaffected by spironolactone, whereas the STZ+SPL group showed considerable improvement in urine albumin and podocyte excretion, as well as up-regulation of integrin alpha 3. Our results suggest that spironolactone ameliorates impaired podocytic adhesion capacity and prevents STZ-induced DN progression.

Effect of ovarian stimulation with human menopausal gonadotropin and recombinant follicle stimulating hormone on the expression of integrins alpha3, beta1 in the rat endometrium during the implantation period.

OBJECTIVE: To investigate the effect of exogenous ovarian stimulation with human menopausal gonadotropin (hMG) and recombinant follicle stimulating hormone (rFSH) on the expression of integrins alpha(3), beta(1) in the rat endometrium during implantation. STUDY DESIGN: Following three successive normal estrous cycles the animals were divided into five groups: Group I (n=10, control group) received no medication; Group II (n=10) received 10 units of hMG; Group III (n=10) received 20 units of hMG; Group IV (n=10) received 10 units of rFSH; Group V (n=10) received 20 units of rFSH at midday of middiestrous. The rats were then mated with fertile males. The animals were sacrificed on the day of implantation. The uterine horns were placed in fixative and paraffin blocks of the tissue were cut in 5 microm sections. The tissues were stained with primary antibodies; monoclonal anti-integrin alpha(3) and monoclonal anti-integrin beta(1) using immunohistochemical methods. The staining intensities of alpha(3) and beta(1) integrins were calculated separately for epithelium and stroma in each group. RESULTS: Staining intensities of alpha(3) and beta(1) integrins in both the epithelium and the stroma were significantly lower in the treatment groups than the control group (p<0.05). CONCLUSION: Ovarian stimulation by low and high doses of HMG and rFSH may have an effect on endometrial receptivity, possibly via a decrease in expression of integrins in the endometrium during the implantation period.

Astragaloside IV improves high glucose-induced podocyte adhesion dysfunction via alpha3beta1 integrin upregulation and integrin-linked kinase inhibition.

Impaired podocyte adhesion to glomerular basement membrane (GBM) may contribute to podocyte detachment from GBM, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we examined the effects of Astragaloside IV (AS-IV), a saponin purified from Astragalus membranaceus (Fisch) Bge, on high glucose-induced cell adhesion dysfunction in cultured mouse podocytes. Cells were seeded into 96-well plates coated with basement membrane protein complex (BMC). The cells were incubated for 12h in media containing 30 mM glucose (HG) with 10, 50 and 100 microg/ml of AS-IV. The cells were also exposed to HG media with 100 microg/ml of AS-IV for 3, 6, 12 and 24h. Cell adhesion assays were performed by fluorescence and centrifugation methods, respectively. Levels of mRNA were determined by quantitative reverse transcriptase real-time PCR and protein expression was analyzed by immunoblotting. HG strongly inhibited adhesion of podocytes to BMC, accompanied by reduction in alpha(3)beta(1) integrin mRNA and protein expression, as well as increase in integrin-linked kinase (ILK) activity and expression. When podocytes under HG stimulation were treated with AS-IV, a dose- and time-dependent increase in cell-matrix adhesion was observed, which was significant from 10 microg/ml of AS-IV and from 6h of incubation of AS-IV with 100 microg/ml. This was accompanied by significant increases in alpha(3)beta(1) integrin mRNA and protein expression, as well as inhibition of ILK activation and overexpression. These results suggest that AS-IV improve HG-induced podocyte adhesion dysfunction, which is partly attributed to alpha(3)beta(1) integrin upregulation and ILK inhibition.

Endometriosis is characterized by an impaired localization of laminin-5 and alpha3beta1 integrin receptor.

Endometriosis is an estrogen-correlated benign disease characterized by a marked ability of endometrial-like cells to invade and proliferate outside uterine cavity, resembling for some invasive aspect the cancer growth. The molecular mechanisms regulating endometrial cell invasiveness are mostly unknown, although interactions between extracellular matrix (ECM) proteins and their transmembrane receptors, integrins, are likely to play a central role. In particular, laminin (Ln)-5 could be closely involved, as it is in cancer. We have investigated the expression of Ln-1, Ln-5, and collagen IV (Coll IV) ECM proteins and their receptors, alpha3beta1 and alpha6beta4 integrins, in atrophic, proliferative, and secretive endometrium and in endometriosis. The results show that Ln-5, but not Ln-I and Coll IV, is altered in secretive endometrium as well as in endometriosis tissues. No alterations are observed in atrophic or proliferative endometrium. Consistently, the polarization of both integrin subunits alpha3 and beta1, but not alpha6 and beta4, is altered in secretive endometrium and endometriosis tissues, but not in atrophic and proliferative endometrium. These results seem to suggest that Ln-5 and alpha3beta1 could be involved in the invasive mechanism of endometriosis. The altered expression of Ln-5, by upregulating matrix metalloproteases activity, suggest an invading process similar to that of many cancer processes.

Spatial and temporal control of laminin-332 (5) and -511 (10) expression during induction of anagen hair growth.

Basement membrane plays important roles in hair growth. We characterized changes in laminin isoform expression during hair cycling. At the mRNA level, laminin-511 (10) expression underwent a steady increase during anagen stages. In contrast, laminin-332 (5) expression was initially upregulated in outer root sheath (ORS) keratinocytes at anagen II and then transiently downregulated. Laminin-332 significantly increased coincident with the signal in inner root sheath and hair matrix cells after anagen IV. Levels of laminin-332 proteins were also upregulated at late anagen I-III but dropped after anagen IV. This decrease coincided with increased levels of mRNA encoding the two proteases, membrane type 1 metalloproteinase and bone morphogenetic protein 1, involved in laminin-332 processing. Immunohistochemistry demonstrated that laminin-332 and alpha6 beta4 integrin were well colocalized, but their signals were remarkably decreased in the lower half of follicles after anagen VI. Consistent with these data, ultrastructurally mature hemidesmosomes were seen in ORS keratinocytes at anagen II, whereas at anagen VI, only fragmental hemidesmosomes were present. In hair follicle culture, laminin-511 (10)/521 (11)-rich human placental laminin enhanced hair growth, whereas recombinant laminin-332 antagonized hair growth induced by laminin-511. Our results indicate a positive role for laminin-511 and a negative role for laminin-332 on hair growth.

Increased expression of integrin alpha3beta1 in highly brain metastatic subclone of a human non-small cell lung cancer cell line.

To clarify the roles of integrin and extracellular matrix (ECM) in the process of non-small cell lung cancer (NSCLC) brain metastasis, we established an in vivo model of brain metastasis of human NSCLC cell line EBC-1/original in athymic mice, and established highly brain metastatic subclone EBC-1/brain and highly bone metastatic subclone EBC-1/bone. Integrin expression of these subclones was evaluated by flow cytometry. In vitro cell attachment, migration and proliferation assays with ECMs were performed using these subclones. Expression of integrin alpha3 subunit was higher in EBC-1/brain than in both EBC-1/original and EBC-1/bone. In vitro cell attachment, migration, and proliferation assays revealed that EBC-1/brain had higher affinity and higher reactivity to laminin than EBC-1/original and EBC-1/bone. Blocking of integrin alpha3beta1 significantly (P < 0.05) decreased brain metastasis by EBC-1/brain. Interaction of integrin alpha3beta1 and laminin plays important roles in the process of brain metastasis of non-small cell lung cancer.

Quantification of thrombospondin-1 secretion and expression of alphavbeta3 and alpha3beta1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells.

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface alphavbeta3 and alpha3beta1 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of alphavbeta3 and alpha3beta1 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of alphavbeta3 and alpha3beta1 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/l x 10(6) cells/24 h (mean +/- SD = 626 +/- 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 microg. Seven of eight non-glioma cell lines secreted less than 100 ng of TSP-1. All glioma cell lines expressed alpha3beta1 integrin and syndecan-1, and seven of 10 glioma cell lines expressed alphavbeta3 integrin. Treatment of the glioma cell lines with TGF-beta2 did not change the expression of alphavbeta3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with alphavbeta3 and alpha3beta1 integrins, and syndecan-1.

Keratinocyte motility induced by TGF-beta1 is accompanied by dramatic changes in cellular interactions with laminin 5.

Transforming growth factor-beta1 (TGF-beta1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF-beta1 induced a marked decrease in their adhesion capacity to processed alpha3-containing laminin 5-coated surfaces. Indeed, the expression levels of alpha3 and alpha6 integrin subunit mRNA and protein, as well as the cell surface alpha3beta1 and alpha6beta4 integrins, were down-regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF-beta1 induced migrating keratinocytes is present in its unprocessed form [Décline and Rousselle, 2001: J. Cell Sci. 114:811-823]. We show here that TGF-beta1 treatment of the cells promoted a significant increase in their adhesion to the alpha3 chain carboxy-terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that alpha6beta4 and alpha3beta1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the alpha3 LG4/5 module present on unprocessed laminin 5.

Induction of a laminin isoform and alpha(3)beta(1)-integrin in renal ischemic injury and repair in vivo.

Ischemic injury to the kidney, a major cause of acute renal failure, leads to the detachment and loss of numerous tubular epithelial cells. Integrin-laminin interactions may promote regeneration of the damaged epithelium by influencing kidney epithelial cell adhesion and differentiation. Laminins are major structural components of basement membranes. Of the various laminin isoforms, laminin-5 is of particular interest because of its proposed role in the healing of skin wounds. In this study, we investigate the expression of laminin-5 in rat kidney after unilateral ischemia. Using a polyclonal antibody generated against laminin-5, we find that immunostaining is confined to the basement membranes of collecting ducts in the papilla and the major and minor calyces in normal kidney. With injury and regeneration, however, immunostaining becomes much more intense and widespread in basement membranes along the nephron. Immunoblotting of ischemic kidney extracts reveals significantly increased expression of a polypeptide of approximately 220 kDa, possibly corresponding to a precursor of one of the three laminin-5 chains. Immunoblotting and immunostaining also demonstrate significantly increased expression and altered localization of the alpha(3)-integrin subunit, a receptor for laminin-5. These results indicate that there is induction of a laminin isoform, possibly laminin-5, and alpha(3)beta(1)-integrin in the ischemic kidney and may implicate this receptor-ligand combination in the pathogenesis of acute renal failure and/or repair of the injured kidney epithelium.

Distribution of laminin 5, integrin receptors, and branching morphogenesis during human fetal lung development.

The role of the epithelial adhesion ligand laminin 5 (LN5) in lung development has been poorly investigated. To determine its potential involvement in lung organogenesis, we used immunofluorescence microscopy to investigate the distribution of LN5 and its integrin (Int) receptors alpha2beta1, alpha3beta1, alpha6beta1, and alpha6beta4 during human fetal airway branching morphogenesis and respiratory epithelium differentiation. At the pseudoglandular and canalicular stages of airway development, LN5 and its constituent chains were localized in the basement membrane (BM) of the proximal respiratory tubules and in the cytoplasm of the epithelial cells forming the growing epithelial buds, which expressed Int alpha2beta1, alpha3beta1, and, transiently, alpha6beta1. At the alveolar and adult stages, LN5 and its constituent chains were localized both in the BM of evolving and differentiated bronchioles and in the alveolar parenchyma. The bronchiolar epithelium markedly expressed Int alpha2beta1 and alpha3beta1, whereas the alveolar parenchyma strongly expressed Int alpha2beta1, alpha3beta1, and alpha6beta1. Throughout fetal development and in the adult, LN5 and its constituent chains were detected both in the tracheal BM, regardless of the degree of epithelial differentiation, and in the cytoplasm of the cells at the invading front of the growing glandular ducts. Ultrastructural studies showed that nucleation of the hemidesmosomes (HDs) correlated with the differentiation of the tracheal epithelium. These results suggest that LN5 may play multiple roles during branching morphogenesis, by modulating proliferation and/or migration of the epithelial cells in the respiratory buds and by establishing branch points, through interaction initially with Int alpha6beta1 and later with Int alpha2beta1 and alpha3beta1. We also propose that LN5 may regulate the differentiation of the tracheal epithelium by means of Int-beta4, which governs HD nucleation.