Downregulation of ITGA6 confers to the invasion of multiple myeloma and promotes progression to plasma cell leukaemia.

BACKGROUND: Secondary plasma cell leukaemia (sPCL) is an aggressive form of multiple myeloma (MM), but the mechanism underlying MM progresses into PCL remains unknown. METHODS: Gene expression profiling of MM patients and PCL patients was analysed to identify the molecular differences between the two diseases. Cox survival regression and Kaplan-Meier analysis were performed to illustrate the impact of integrin subunit alpha 6 (ITGA6) on prognosis of MM. Invasion assays were performed to assess whether ITGA6 regulated the progression of MM to PCL. RESULTS: Gene expression profiling analyses showed that cell metastasis pathways were enriched in PCL and ITGA6 was differentially expressed between PCL and MM. ITGA6 expression was an independent prognostic factor for event-free survival (EFS) and overall survival (OS) of MM patients. Moreover, the stratification ability of the International Staging System (ISS) of MM was improved when including ITGA6 expression. Functional studies uncovered that increased ITGA6 reduced the myeloma cell invasion. Additionally, low expression of ITGA6 resulted from epigenetic downregulating of its anti-sense non-coding RNA, ITGA6-AS1. CONCLUSION: Our data reveal that ITGA6 gradually decreases during plasma cell dyscrasias progression and low expression of ITGA6 contributes to myeloma metastasis. Moreover, ITGA6 abundance might help develop MM prognostic stratification.

Regulation of integrin α6A by lactogenic hormones in rat pancreatic ß-cells: Implications for the physiological adaptation to pregnancy.

AIM: During pregnancy, the maternal ß-cell mass is increased in order to adapt to the physiological changes in insulin demand. Lactogenic hormones stimulate rodent ß-cell attachment and proliferation in vitro. The aim of this study was to identify adhesion molecules involved in expansion of the ß-cell mass during pregnancy in the rat. METHODS: Quantitative RT-PCR was used to evaluate the expression of several integrins and laminins in isolated neonatal rat islets in response to growth hormone (GH) and prolactin (PRL) treatment. Double-immunofluorescence staining of rat pancreas was used to localize the expression of integrin α6ß1. ß-cell proliferation was evaluated by incorporation of bromodeoxyuridine (BrdU). The role of STAT5 phosphorylation was tested by addition of STAT5 mutants. RESULTS: We found that the mRNA level of integrin-α6A, was upregulated 2.5-fold by PRL or GH. During pregnancy, a biphasic 3.4-4.5-fold increase of integrin-α6A and B mRNA levels was detected. A disintegrin peptide (DP) reduced the hormone-stimulated mitotic activity in neonatal rat ß-cells from 2.9 ± 0.4-fold to 1.3 ± 0.3-fold. The hormone-induced expression of α6ß1 integrin was shown to be mediated via STAT5 as a dominant negative (DN) mutant prevented and a constitutive active (CA) mutant augmented the hGH-stimulated expression. The DP was found to inhibit hGH-induced transactivation of the PRL receptor promoter 1A and reduce the hGH-induced phosphorylation of STAT5. CONCLUSION: These results show that integrin-α6 in ß-cells is upregulated by lactogenic hormones and is required but not sufficient for the expansion of the ß-cell mass in pregnancy in the rat, which may have implications for the understanding and treatment of gestational diabetes mellitus.

Laminin-binding integrins are essential for the maintenance of functional mammary secretory epithelium in lactation.

Integrin dimers α3/ß1, α6/ß1 and α6/ß4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation.

Integrin α6 (CD49f), The Microenvironment and Cancer Stem Cells.

Cancer is a highly prevalent and potentially terminal disease that affects millions of individuals worldwide. Here, we review the literature exploring the intricacies of stem cells bearing tumorigenic characteristics and collect evidence demonstrating the importance of integrin α6 (ITGA6, also known as CD49f) in cancer stem cell (CSC) activity. ITGA6 is commonly used to identify CSC populations in various tissues and plays an important role sustaining the self-renewal of CSCs by interconnecting them with the tumorigenic microenvironment.

Physiological stretch induced proliferation of human urothelial cells via integrin α6-FAK signaling pathway.

AIMS: To test a kind of stretch pattern which is the optimum stress parameter to promote human urothelial cells (HUCs) proliferation, and to investigate the roles of integrin subunits and their pathway in the HUCs proliferation induced by physiological stretch. METHODS: HUCs were seeded on silicone membrane, and subjected to four kinds of stretch (0,5%,10%,15% elongation) for 24 h, as controlled by a BioDynamic® bioreactor. Cell proliferation, viability and cycle distribution were examined using Cell Counting Kit-8 and flow cytometry, respectively. The gene and protein expression of integrin subunits and focal adhesion kinase (FAK) in each group were assessed by Real-time PCR(RT-PCR) and western blot, respectively. Small interfering RNAs (siRNA) were applied to knockdown integrin α6 and FAK expression in HUCs, and FAK inhibitor was used to validate the role of α6 and FAK in cell proliferation under physiological stretch. RESULTS: The proliferation of HUCs were highest in the 5% elongation group compared to static control, 10% and 15% elongation group. RT-PCR and western blot showed that 5% cyclic stretch significantly promoted the expression of integrin α6 and FAK. The stretch-induced cell proliferation and FAK expression was inhibited by siRNA of integrin α6. Further study with FAK inhibitor revealed that elongation promoted proliferation though integrin α6 and FAK signaling pathway. CONCLUSIONS: Physiological stretch induced HUCs proliferation via integrin α6-FAK signaling pathway, and 5% elongation may be the optimal stress parameter to promote the cell proliferation.

CD34 and CD49f Double-Positive and Lineage Marker-Negative Cells Isolated from Human Myometrium Exhibit Stem Cell-Like Properties Involved in Pregnancy-Induced Uterine Remodeling.

Repeated and dramatic pregnancy-induced uterine enlargement and remodeling throughout reproductive life suggests the existence of uterine smooth muscle stem/progenitor cells. The aim of this study was to isolate and characterize stem/progenitor-like cells from human myometrium through identification of specific surface markers. We here identify CD49f and CD34 as markers to permit selection of the stem/progenitor cell-like population from human myometrium and show that human CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells exhibit stem cell-like properties. These include side population phenotypes, an undifferentiated status, high colony-forming ability, multilineage differentiation into smooth muscle cells, osteoblasts, adipocytes, and chondrocytes, and in vivo myometrial tissue reconstitution following xenotransplantation. Furthermore, CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells proliferate under hypoxic conditions in vitro and, compared with the untreated nonpregnant myometrium, show greater expansion in the estrogen-treated nonpregnant myometrium and further in the pregnant myometrium in mice upon xenotransplantation. These results suggest that the newly identified myometrial stem/progenitor-like cells influenced by hypoxia and sex steroids may participate in pregnancy-induced uterine enlargement and remodeling, providing novel insights into human myometrial physiology.

Tie2-dependent knockout of α6 integrin subunit in mice reduces post-ischaemic angiogenesis.

AIMS: Integrins α6ß1 and α6ß4 are receptors for laminins, the main components of the basement membrane underlying the endothelial cells. In vitro, α6 integrin subunit (α6) expression at the surface of endothelial cells and their progenitors (EPCs) is up-regulated by pro-angiogenic growth factors and is crucial for adhesion, migration, and pseudotube formation. We investigated the role for α6 in post-ischaemic vascular repair in vivo. METHODS AND RESULTS: We used the cre-lox system to generate a mouse line with specific α6 gene deletion in Tie2-lineage cells. In a model of hind-limb ischaemia, Tie2-dependent α6 deletion reduced neovessel formation and reperfusion of the ischaemic limb. Concerning the role for α6 in post-ischaemic vasculogenesis, we showed previously that α6 was required for EPC recruitment at the site of ischaemia. Here, we found that α6 deletion also reduced EPC mobilization from the bone marrow after ischaemia. Examination of the ischaemic muscles showed that Tie2-dependent α6 deletion decreased the recruitment of pro-angiogenic Tie2-expressing macrophages. In the Matrigel plug assay, fibroblast growth factor-2-induced vascularization was diminished in mice lacking endothelial α6. To specifically investigate the role for α6 in angiogenesis, aortic rings were embedded in Matrigel or collagen and cultured ex vivo. In Matrigel, neovessel outgrowth from rings lacking α6 was strongly diminished, whereas no genotype-dependent difference occurred for rings in collagen. CONCLUSION: α6 plays a major role in both post-ischaemic angiogenesis and vasculogenesis.

Effect of busui shengxue granule (see text) on chronic aplastic anemia patients' hematopoietic adhesion molecule VLA-6/CD49f and its ligand laminin.

OBJECTIVE: To observe the effect of Busui Shengxue Granule ((see text) Herbal granule for replenishing marrow to produce blood) on chronic aplastic anemia (CAA) patients' integrin alpha 6 (VLA-6/CD49f) and laminin (Ln). METHODS: Sixty-five patients were divided into experimental group and control group through random number table. There were 34 patients, 17 were male and 17 female, aged 2-67, with a medianage of 30.2 +/- 8.6, in the experimental group, including 17 patients of kidney-yin deficiency and 17 of kidney-yang deficiency, treated by Busui Shengxue Granule. There were 31 patients in the control group, 16 were male and 15 female, aged 4-65, with a medianage of 31.2 +/- 8.0; administered Zaizhang Shengxue Tablet (see text) Herbal tablet for chronic aplastic anemia). Both groups were treated for six months and compared with 10 normal persons after the treatment. Flow cytometry was adopted to detect the change in the expression of VLA-6/CD49f, receptor in mononuclear cells of CAA patients and normal persons. Enzyme-linked immunosorbent assay was applied to detect the expression of peripheral serum Ln. RESULTS: CAA patients' VLA-6/CD49f was in the state of low expression and Ln in the state of high expression. After the treatment, both VLA-6/CD49f and Ln were regulated to some extent and the change in the experimental group was better than that of the control group. Compared with the kidney-yin deficiency patients, those indices of kidney-yang deficiency patients were easier to correct. CONCLUSION: The VLA-6/CD49f and Ln expressions of CAA patients are abnormal. The treatment with Busui Shengxue Granule makes both of them improved.

Integrin subunits alpha5 and alpha6 regulate cell cycle by modulating the chk1 and Rb/E2F pathways to affect breast cancer metastasis.

BACKGROUND: Although integrins have been implicated in the progression of breast cancer, the exact mechanism whereby they exert this regulation is clearly not understood. To understand the role of integrins in breast cancer, we examined the expression levels of several integrins in mouse breast cancer cell lines by flow cytometry and the data were validated by Western and RT-PCR analysis. The importance of integrins in cell migration and cell invasion was examined by in vitro assays. Further the effect of integrins on metastasis was investigated by in vivo experimental metastasis assays using mouse models. RESULTS: Integrin α5 subunit is highly expressed in the nonmetastatic cell line 67NR and is significantly low in the highly invasive cell line 4T1. In contrast, expression levels of integrin α6 subunit are high in 4T1 cells and low in 67NR cells. In vitro data indicated that overexpression of α5 subunit and knockdown of α6 integrin subunit inhibited cell proliferation, migration, and invasion. Our in vivo findings indicated that overexpression of integrin α5 subunit and knockdown of α6 subunit decreased the pulmonary metastasis property of 4T1 cells. Our data also indicated that overexpression of alpha 5 integrin subunit and suppression of alpha6 integrin subunit inhibited cells entering into S phase by up-regulating p27, which results in downregulation of cyclinE/CDK2 complexes, This suggests that these integrins regulate cell growth through their effects on cell-cycle-regulated proteins. We also found that modulation of these integrins upregulates E2F, which may induce the expression of chk1 to regulate cdc25A/cyclin E/CDK2/Rb in a feedback loop mechanism. CONCLUSION: This study indicates that Integrin α5 subunit functions as a potential metastasis suppressor, while α6 subunit functions as a metastasis promoter. The modulation of integrins reduces cdc25 A, another possible mechanism for downregulation of CDK2. Taken together we demonstrate a link between integrins and the chk1-cdc25-cyclin E/CDK2-Rb pathway.

Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis.

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. alpha6beta1- and alpha6beta4-integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial alpha6-integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the alpha6-integrin-subunit when compared with normal breast tissue. These data suggest that a decrease in alpha6-integrin-subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial alpha6-integrin subunit affects tumour growth and angiogenesis, we generated alpha6fl/fl-Tie1Cre+ mice and showed that endothelial deletion of alpha6-integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial alpha6-integrin deficiency elevated significantly VEGF-mediated angiogenesis both in vivo and ex vivo. In particular, alpha6-integrin-deficient endothelial cells displayed increased levels of VEGF-receptor 2 (VEGFR2) and VEGF-mediated downstream ERK1/2 activation. By developing the first endothelial-specific alpha6-knockout mice, we show that the expression of the alpha6-integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo.

Fusion as the result of sperm-somatic cell interaction.

The research has been designed to investigate whether acrosome-reacted spermatozoa can fuse with somatic cells and to check whether this event may involve the molecular machinery implicated in the sperm-egg fusion. Boar spermatozoa were capacitated in vitro and then treated with A23187 to induce acrosome reaction and activate their fusogenic potential. Reacted spermatozoa, loaded with the membrane-permeant fluorescent dye calcein AM, were incubated with plated granulosa cells or cells derived from stable cell lines: CRFK, VERO, and ESK4. The fusion between spermatozoa and somatic cells was revealed by the diffusion of the fluorescent dye from the sperm to the cell as membrane fusion and cytoplasmic continuity between the two cells were established. The involvement of integrin alpha6 and tetraspanin CD9 in the process of fusion was assessed by carrying out the experiment in the presence of antibodies against these molecules. Moreover, the incidence of fusion displayed by the different cell types used was analyzed in relation to their content in the above molecules assessed by western blot and immunostaining. The role of CD9 was additionally investigated by using CD9-negative cells. The data presented demonstrate that boar spermatozoa can fuse with different somatic cell types derived from different species and the process requires the combined presence of both integrin and tetraspanin molecules on the cell plasma membrane.

Flightless I regulates hemidesmosome formation and integrin-mediated cellular adhesion and migration during wound repair.

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.

Overexpression of integrin alpha6 and beta4 enhances adhesion and proliferation of human retinal pigment epithelial cells on layers of porcine Bruch's membrane.

Transplantation of retinal pigment epithelium (RPE) following removal of choroidal neovascular membranes has been attempted in patients with age-related macular degeneration (AMD). However, inability of transplanted RPE to initially attach and subsequently proliferate on Bruch's membrane may lead to failure of RPE transplants and poor visual outcomes. Integrin alpha(6)beta(4) functions as a receptor for laminin, the major component of Bruch's membrane, and mediates the stable attachment of most epithelial cells to the underlying basement membrane. To improve adhesion and proliferation of transplanted RPE on Bruch's membrane, we elucidated the roles of integrin alpha(6)beta(4) in RPE adhesion to extracellular matrix and investigated whether ex vivo gene transfer of integrin alpha(6) and beta(4) in RPE could promote adhesion and proliferation of transplanted RPE on Bruch's membrane. The expression of integrin alpha(6) and beta(4) mRNA and surface protein in ARPE-19 cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis. We generated point mutation in the ligand binding domain of integrin alpha(6) and beta(4) by using site-directed mutagenesis and transfected these mutated constructs into ARPE-19 cells. Adhesion assay was used to determine the roles of integrin alpha(6) and beta(4) in RPE adhesion to extracellular matrix. In addition, we transfected full-length alpha(6) cDNA or beta(4) cDNA into ARPE-19 cells. The reattachment and proliferation ratios of alpha(6)-cDNA- or beta(4)-cDNA-transfected ARPE-19 cells on different layers of Bruch's membrane were determined by cell adhesion and proliferation assays. Cell morphology and surface coverage were evaluated by scanning electron microscopy 7 days after plating on various layers of Bruch's membrane. We found that integrin alpha(6) and beta(4) mRNA and proteins were constitutively expressed in ARPE-19 cells. Decreased endogenous integrin alpha(6) and beta(4) expression by selective mutation of amino acid residues caused a significant reduction in adhesion of ARPE-19 cells to laminin 5. Modification of integrin expression by transfection of alpha(6) cDNA into ARPE-19 cells induced a significant increase in cell adhesion to laminin 5, fibronectin, whereas transfection with beta(4) cDNA caused increased adhesion only to laminin 5. alpha(6)-cDNA-transfectants increased cell attachment and proliferation on all layers of Bruch's membrane, whereas beta(4)-cDNA-transfectants enhanced adhesion and proliferation on basal lamina and inner collagenous layers. These data indicate that integrin alpha(6) and beta(4) play a role in adhesion of ARPE-19 cells to extracellular matrix. Modification of integrin expression by ex vivo genetic manipulation in RPE might be an alternative strategy to increase the success of RPE transplantation.

Role of alpha 6 integrin subunit in branching morphogenesis of fetal mouse submandibular gland: investigation by mesenchyme-free epithelial culture system.

The submandibular gland (SMG) of the fetal mouse is a well-studied model for the epithelial-mesenchymal interactions required for branching morphogenesis (BrM), which involves cleft formation and stalk elongation. In a previous report, we showed that alpha 6 integrin subunit is involved in BrM of this gland rudiment, since the neutralizing antibody against alpha 6 integrin subunit, GoH3, strongly inhibited branching of cultured intact E13 SMG. In this study, we investigated whether GoH3 inhibits cleft formation and/or stalk elongation during BrM of cultured mesenchyme-free epithelial rudiments of fetal SMG and also analyzed by Western blotting the levels of phosphorylation of ERK1/2, which is a signaling molecule known to regulate BrM of the fetal mouse SMG.

The role of alpha 6 integrin in prostate cancer migration and bone pain in a novel xenograft model.

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain.

CD151 accelerates breast cancer by regulating alpha 6 integrin function, signaling, and molecular organization.

CD151, a master regulator of laminin-binding integrins (alpha(6)beta(4), alpha(6)beta(1), and alpha(3)beta(1)), assembles these integrins into complexes called tetraspanin-enriched microdomains. CD151 protein expression is elevated in 31% of human breast cancers and is even more elevated in high-grade (40%) and estrogen receptor-negative (45%) subtypes. The latter includes triple-negative (estrogen receptor, progesterone receptor, and HER2 negative) basal-like tumors. CD151 ablation markedly reduced basal-like mammary cell migration, invasion, spreading, and signaling (through FAK, Rac1, and lck) while disrupting epidermal growth factor receptor (EGFR)-alpha(6) integrin collaboration. Underlying these defects, CD151 ablation redistributed alpha(6)beta(4) integrins subcellularly and severed molecular links between integrins and tetraspanin-enriched microdomains. In a prototypical basal-like mammary tumor line, CD151 ablation notably delayed tumor progression in ectopic and orthotopic xenograft models. These results (a) establish that CD151-alpha(6) integrin complexes play a functional role in basal-like mammary tumor progression; (b) emphasize that alpha(6) integrins function via CD151 linkage in the context of tetraspanin-enriched microdomains; and (c) point to potential relevance of CD151 as a high-priority therapeutic target, with relative selectivity (compared with laminin-binding integrins) for pathologic rather than normal physiology.

Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling.

Laminin alpha chains have unique spatiotemporal expression patterns during development and defining their function is necessary to understand the regulation of epithelial morphogenesis. We investigated the function of laminin alpha5 in mouse submandibular glands (SMGs). Lama5(-/-) SMGs have a striking phenotype: epithelial clefting is delayed, although proliferation occurs; there is decreased FGFR1b and FGFR2b, but no difference in Lama1 expression; later in development, epithelial cell organization and lumen formation are disrupted. In wild-type SMGs alpha5 and alpha1 are present in epithelial clefts but as branching begins alpha5 expression increases while alpha1 decreases. Lama5 siRNA decreased branching, p42 MAPK phosphorylation, and FGFR expression, and branching was rescued by FGF10. FGFR siRNA decreased Lama5 suggesting that FGFR signaling provides positive feedback for Lama5 expression. Anti-beta1 integrin antibodies decreased FGFR and Lama5 expression, suggesting that beta1 integrin signaling provides positive feedback for Lama5 and FGFR expression. Interestingly, the Itga3(-/-):Itga6(-/-) SMGs have a similar phenotype to Lama5(-/-). Our findings suggest that laminin alpha5 controls SMG epithelial morphogenesis through beta1 integrin signaling by regulating FGFR expression, which also reciprocally regulates the expression of Lama5. These data link changes in basement membrane composition during branching morphogenesis with FGFR expression and signaling.

Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay.

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP-KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow-derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of beta1 and alpha6 integrins and are distinct from SP-KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP-KCs and integrin-bright keratinocytes, we could not document their superior long-term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP-KCs and integrin-bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different human major histocompatibility (MHC) class I antigens were grafted onto immunocompromised mice, and the intrinsic MHC class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP-KCs showed little competitive expansion in vivo and were not enriched for KSCs. In contrast, keratinocytes expressing elevated levels of alpha6 integrin and low levels of CD71 (alpha6-bright/CD71-dim) expanded over 200-fold during the 33-week in vivo study. These results definitively demonstrate that human alpha6-bright/CD71-dim keratinocytes are enriched with KSCs, whereas SP-KCs are not.