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PubMed2006

Rac1 is crucial for hair follicle integrity but is not essential for maintenance of the epidermis.

Rac1 is a small GTPase that regulates the actin cytoskeleton but also other cellular processes. To investigate the function of Rac1 in skin, we generated mice with a keratinocyte-restricted deletion of the rac1 gene. Rac1-deficient mice lost nearly all of their hair within a few weeks after birth. The nonpermanent part of mutant hair follicles developed constrictions; lost expression of hair follicle-specific keratins, E-cadherin, and alpha6 integrin; and was eventually removed by macrophages. The permanent part of hair follicles and the sebaceous glands were maintained, but no regrowth of full-length hair follicles was observed. In the skin of mutant mice, epidermal keratinocytes showed normal differentiation, proliferation, cell-cell contacts, and basement membrane deposition, demonstrating no obvious defects of Rac1-deficient epidermis in vivo. In vitro, Rac1-null keratinocytes displayed a strong spreading defect and slightly impaired adhesion. These data show that Rac1 plays an important role in sustaining the integrity of the lower part of hair follicles but not in maintenance of the epidermis.

Animals↗
PubMed1998

Loss of laminin and of the laminin receptor integrin subunit alpha 6 in situ correlates with cytokine induced down regulation of alpha 6 on fibroblast-like synoviocytes from rheumatoid arthritis.

OBJECTIVE: To investigate in situ the expression of the integrin receptor subunits alpha 6 and beta 1 and the distribution of the ligand laminin in the synovia from osteoarthritis (OA) and rheumatoid arthritis (RA) patients and to study the effect of cytokines and antirheumatic drugs on the expression of the alpha 6 and beta 1 integrin subunits on long term cultures of fibroblast-like synoviocytes (FBS) derived from OA and RA. METHODS: The expression of the alpha 6 and beta 1 integrin subunits and the distribution of laminin were examined immunohistochemically in normal synovia and in synovia from patients with OA and RA. The effect of proinflammatory cytokines (IL1 beta and TNF alpha), and of antirheumatic drugs (salicylic acid, dexamethasone, and methotrexate) on the alpha 6 and beta 1 expression of cultured normal FBS and FBS from patients with OA and RA was determined by flow cytometry. RESULTS: In normal synovia and in OA synovia samples with a low grade of inflammation, synovial lining cells (SLC) showed a parallel expression and distribution of alpha 6 and laminin. In synovia samples of OA with a higher grade of inflammation and in the majority of RA synovia samples laminin was pericellularly distributed in a low number of SLC, whereas alpha 6 was expressed on the surface of a high number of SLC. In RA synovia samples with severe inflammatory changes the gradual loss of laminin generally corresponded to a decrease of the alpha 6 integrin subunit. beta 1 was always strongly expressed in all synovia samples detected. Proinflammatory cytokines up regulated the expression of alpha 6 and beta 1 on OA-FBS, whereas these effectors decreases alpha 6 and beta 1 on RA-FBS. In contrast, antirheumatic drugs, in particular methotrexate and dexamethasone, reduced the expression of alpha 6 and beta 1 on OA-FBS, whereas the same treatment on RA-FBS stimulated the expression of these integrin subunits. CONCLUSION: The gradual loss of laminin in chronic synovitis may contribute to the altered expression of alpha 6 in SLC. IL1 beta and TNF alpha down regulated the expression of the alpha 6 and beta 1 integrin subunits on long term cultures of FBS derived from RA. Therefore, these cytokines may be among the effectors regulating the expression of the alpha 6 integrin subunit in SLC in vivo. As antirheumatic drugs increase the expression of alpha 6 on RA-FBS, the presence of the laminin receptor may confer a protective effect on the synovia in vivo.

Antigens, CD↗
PubMed2006

Two populations of Thy1-positive mesenchymal cells regulate in vitro maturation of hepatic progenitor cells.

We previously reported that the in vitro maturation of CD49f(+)Thy1(-)CD45(-) (CD49f positive) fetal hepatic progenitor cells (HPCs) is supported by Thy1-positive mesenchymal cells derived from the fetal liver. These mesenchymal cell preparations contain two populations, one of a cuboidal shape and the other spindle shaped in morphology. In this study, we determined that the mucin-type transmembrane glycoprotein gp38 could distinguish cuboidal cells from spindle cells by immunocytochemistry. RT-PCR analysis revealed differences between isolated CD49f(+/-)Thy1(+)gp38(+)CD45(-) (gp38 positive) cells and CD49f(+/-)Thy1(+)gp38(-)CD45(-) (gp38 negative) cells, whereas both cells expressed mesenchymal cell markers. The coculture with gp38-positive cells promoted the maturation of CD49f-positive HPCs, which was estimated by positivity for periodic acid-Schiff (PAS) staining, whereas the coculture with gp38-negative cells maintained CD49f-positive HPCs negative for PAS staining. The expression of mature hepatocyte markers, such as tyrosine aminotransferase, tryptophan-2,3-dioxygenase, and glucose-6-phosphatase, were upregulated on HPCs by coculture with gp38-positive cells. Furthermore, transmission electron microscopy revealed the acquisition of mature hepatocyte features by HPCs cocultured with gp38-positive cells. This effect on maturation of HPCs was inhibited by the addition of conditioned medium derived from gp38-negative cells. By contrast, the upregulation of bromodeoxyuridine incorporation by HPCs demonstrated the proliferative effect of coculture with gp38-negative cells. In conclusion, these results suggest that in vitro maturation of HPCs promoted by gp38-positive cells may be opposed by an inhibitory effect of gp38-negative cells, which likely maintain the immature, proliferative state of HPCs.

Actins↗
PubMed2004

Altered expression of metastasis-associated and regulatory molecules in effusions from breast cancer patients: a novel model for tumor progression.

PURPOSE: The aim of this study was to characterize phenotypic alterations along the progression of breast carcinoma from primary tumor to pleural effusion through analysis of the expression of proteases, laminin receptors (LRs), and transcription factors involved in invasion and metastasis. EXPERIMENTAL DESIGN: The material studied consisted of 60 malignant pleural effusions from breast cancer patients and 68 corresponding solid tumors (37 primary and 31 metastatic tumors). Expression of matrix metalloproteinases [MMPs (MMP-1, MMP-2, MMP-9, and MMP-14)], the MMP inhibitor tissue inhibitor of metalloproteinase-2, the MMP inducer EMMPRIN, the 67-kDa LR, the alpha6 integrin subunit, and the transcription factors AP-2, Ets-1, and PEA3 was studied using immunohistochemistry, mRNA in situ hybridization, reverse transcription-polymerase chain reaction, zymography, and flow cytometry. Hormone receptor (estrogen receptor and progesterone receptor) status and c-erbB-2 status were also studied. RESULTS: Significantly reduced estrogen receptor (P < 0.001) and progesterone receptor (P = 0.001) expression was seen in effusions compared with primary tumors, with opposite findings for c-erbB-2 (P = 0.003). Tumor cell MMP-2 protein expression in effusions was higher than that in primary tumors (P < 0.001) and lymph node metastases (P = 0.01). In situ hybridization demonstrated higher MMP-2 (P = 0.007), PEA3 (P = 0.038), and EMMPRIN (P = 0.026) mRNA expression in effusions. The time to progression from primary tumor to effusion was significantly shorter for patients whose primary tumors expressed MMP-1 (P = 0.016) and who expressed the 67-kDa LR protein in primary tumor (P = 0.007) and effusion (P = 0.015). CONCLUSIONS: Our data provide documented evidence of molecular events that occur during the progression of breast carcinoma from primary tumor to effusion. The coordinated up-regulation of MMP-2 and Ets transcription factors in carcinoma cells in effusions is in full agreement with our previous reports linking these factors to poor prognosis in ovarian cancer. The rapid progression to effusion in cases showing MMP-1 and 67-kDa LR expression in primary tumor cells links aggressive clinical behavior with expression of metastasis-associated molecules in this setting.

Adult↗
PubMed2002

Identification of highly expressed genes in peripheral blood T cells from patients with atopic dermatitis.

BACKGROUND: Analysis of genes that are differentially expressed in patients with atopic dermatitis (AD) and normal individuals will provide important information on the underlying molecular pathogenetic mechanisms of AD. METHODS: Transcript of freshly isolated peripheral blood T cells from 59 individuals were analyzed with a fluorescent differential display (FDD) method. Ninety-two differentially expressed genes were identified in this manner. Additionally, real-time quantitative RT-PCR was employed to investigate the expression of the FDD-selected genes and also genes related to T cell function. RESULTS: A number of genes, including CC chemokine receptor 4, T cell-specific tyrosine kinase (Emt/Itk), integrin beta1, integrin alpha6, IQGAP1 and MAR/SAR DNA-binding protein (SATB1), were shown to be more highly expressed in patients with moderate and/or severe AD than in controls or patients with mild AD. Because the products of these upregulated genes influence chemotaxis, adhesion, migration and Th2 polarization, it is suggested that in more severe AD, circulating T cells may function differently in this regard. Several other genes, the role of which in T cell function is currently unknown, were also found to be differentially expressed in AD. These included the heat shock protein 40 and vasopressin-activated calcium-mobilizing receptor 1. CONCLUSION: The upregulated genes identified in this work may serve as useful markers for moderate to severe AD as opposed to normal or mild AD and also as markers indicating progression to more severe AD. Further functional characterization will provide a better understanding of the pathophysiology of circulating T cells in AD.

Adolescent↗
PubMed1996

Granulosa cells express integrin alpha 6: possible involvement of integrin alpha 6 in folliculogenesis.

To identify differentiation antigens of granulosa cells, we have raised two monoclonal antibodies against porcine granulosa cells (POG-2 antibody) and human granulosa cells (OG-1 antibody). Analysis of N-terminal amino acid sequences of purified OG-1 and POG-2 antigens revealed that they were identical to human integrin alpha 6 and its porcine homolog, respectively. An immunohistochemical study showed that integrin alpha 6 was highly expressed on granulosa cells in preovulatory follicles in the human ovary, whereas it was highly expressed on granulosa cells in small follicles in the porcine ovary. As a common characteristic of integrin alpha 6 expression in both ovaries, granulosa cells located in inner layers, which are not in contact with the basal lamina, expressed integrin alpha 6 on the cell surface. This expression profile suggests that the interaction between integrin and extracellular matrices occurs in the granulosa cells located in inner layers. This cell surface interaction is reported to modulate soluble signals to the cells. Since the expression of integrin alpha 6 on inner-layer granulosa cells is related to stages of differentiation, this interaction may be involved in folliculogenesis.

Animals↗
PubMed1997

Rapid disruption of an astrocyte interaction with the extracellular matrix mediated by integrin alpha 6 beta 4 during focal cerebral ischemia/reperfusion.

BACKGROUND AND PURPOSE: Integrins participate in cerebral microvascular integrity and signaling during focal ischemia/ reperfusion. The integrin subunits alpha 1, alpha 6, and beta 1 are distributed identically on normal cerebral microvessels. Studies in epithelium indicate that integrin alpha 6 beta 4, which interacts with laminin-5 in the basal lamina/extracellular matrix, is unique. This study describes the exact location of alpha 6, beta 4, and alpha 6 beta 4 and that their responses in focal cerebral ischemia are relevant to astrocyte-matrix interactions. METHODS: The effect of middle cerebral artery occlusion and subsequent reperfusion on the microvascular expression of alpha 6 beta 4 and laminin-5 in regions of cellular injury (dUTP incorporation) was examined in 15 nonhuman primates. Well-characterized antibodies against human alpha 6, beta 4, alpha 6 beta 4, laminin-5 and laminin-1, endothelial CD31, and vascular markers were measured with computerized video imaging and laser confocal microscopy. RESULTS: Integrin alpha 6 beta 4 was localized on astrocytes where it connects with the extracellular matrix at the astrocyte-vessel interface. It represented 59.3 +/- 16.4% of alpha 6 antigen in cerebral microvessels < 100 microns in diameter. By 2 hours of ischemia, the significant reduction in alpha 6 expression (2P < .001) was accompanied by decreases in beta 4/laminin-5 (0.76 +/- 0.03 to 0.20 +/- 0.09; 2P = .001) and alpha 6 beta 4/laminin-5 (0.73 +/- 0.18 to 0.25 +/- 0.11; 2P = .001) in the region of dUTP incorporation. Parallel changes in laminin-5 and laminin-1 were less pronounced and coincided by 24 hours. CONCLUSIONS: This is the first description of a potential role of integrin alpha 6 beta 4 in the brain, where it mediates astrocyte-matrix interactions. The dramatic disappearance of alpha 6 beta 4 relative to its ligands reflects early loss of integrity between the astrocyte and the vessel wall in selected microvessels in response to ischemia.

Animals↗
PubMed2006

Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane.

PURPOSE: To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM. METHODS: Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin alpha6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography. RESULTS: All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM. CONCLUSIONS: After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.

Amnion↗
PubMed2001

Involvement of T-lymphocytes in periodontal disease and in direct and indirect induction of bone resorption.

Periodontal disease is a peripheral infection involving species of gram-negative organisms. T-lymphocytes can be found in the dense inflammatory infiltrate in this disease. CD4+ and CD8+ T-cells are present in periodontal lesions, as are memory/activated T-lymphocytes. In addition, Th1- and Th2-type T-lymphocytes and their associated cytokines with a subtle polarization to Th1 may be present. Th1-type T-cells up-regulate the production of pro-inflammatory cytokines IL-1 and TNF-alpha, which can induce bone resorption indirectly by promoting differentiation of osteoclast precursors and subsequently by activating osteoclasts. Such osteoclast differentiation is dependent on stimulation of osteoprotegerin ligand (OPG-L) production by osteoblastic cells. By contrast, activated T-cells, by virtue of direct production and expression of OPG-L, can directly promote osteoclast differentiation. OPG-L appears to be predominantly expressed on Th1-type cells. The direct and indirect T-cell involvement in periodontal bone resorption appears to be dependent on the degree of Th1-type T-cell recruitment into inflamed gingival tissues. This T-cell recruitment is regulated by adhesion molecules and chemokines/chemokine receptors. The adhesion molecules involved include alpha4 and alpha6 integrins, LFA-1, and ICAM-1. The Th1-type T-cells preferentially express CCR5 and CXCR3, which are found prominently in diseased gingivae. By contrast, little CCR4, expressed by Th2-type T-cells, can be detected. Also, the chemokine ligands RANTES, MIP1-alpha (both CCR5), and IP-10 (CXCR3 ligand) were elevated in inflamed periodontal tissues. The T-cell features in diseased periodontal tissues can be compared with those in rheumatoid arthritis, wherein bone resorption often attributed to Th1-type T-cell involvement has also been demonstrated.

Alveolar Bone Loss↗
PubMed2006

The metastasis-associated 67-kDa laminin receptor is involved in G-CSF-induced hematopoietic stem cell mobilization.

The 67-kDa laminin receptor (67LR) is a nonintegrin cell-surface receptor with high affinity for laminin, which plays a key role in tumor invasion and metastasis. We investigated the role of 67LR in granulocyte colony-stimulating factor (G-CSF)-induced mobilization of CD34+ hematopoietic stem cells (HSCs) from 35 healthy donors. G-CSF-mobilized HSCs, including CD34+/CD38- cells, showed increased 67LR expression as compared with unstimulated marrow HSCs; noteworthy, also, is the fact that the level of 67LR expression in G-CSF-mobilized HSCs correlated significantly with mobilization efficiency. During G-CSF-induced HSC mobilization, the expression of laminin receptors switched from alpha6 integrins, which mediated laminin-dependent adhesion of steady-state human marrow HSCs, to 67LR, responsible for G-CSF-mobilized HSC adhesion and migration toward laminin. In vitro G-CSF treatment, alone or combined with exposure to marrow-derived endothelial cells, induced 67LR up-regulation in marrow HSCs; moreover, anti-67LR antibodies significantly inhibited transendothelial migration of G-CSF-stimulated marrow HSCs. Finally, G-CSF-induced mobilization in mice was associated with 67LR up-regulation both in circulating and marrow CD34+ cells, and anti-67LR antibodies significantly reduced HSC mobilization, providing the first in vivo evidence for 67LR involvement in stem-cell egress from bone marrow after G-CSF administration. In conclusion, 67LR up-regulation in G-CSF-mobilized HSCs correlates with their successful mobilization and reflects its increase in marrow HSCs, which contributes to the egress from bone marrow by mediating laminin-dependent cell adhesion and transendothelial migration.

ADP-ribosyl Cyclase 1↗
PubMed2001

Endothelial cell activation by myeloblasts: molecular mechanisms of leukostasis and leukemic cell dissemination.

Leukostasis and tissue infiltration by leukemic cells are poorly understood life-threatening complications of acute leukemia. This study has tested the hypothesis that adhesion receptors and cytokines secreted by blast cells play central roles in these reactions. Immunophenotypic studies showed that acute myeloid leukemia (AML) cells (n = 78) of the M0 to M5 subtypes of the French-American-British Cooperative Group expressed various amounts of adhesion receptors, including CD11a, b, c/CD18, CD49d, e, f/CD29, CD54, sCD15, and L-selectin. The presence of functional adhesion receptors was evaluated using a nonstatic adhesion assay. The number of blast cells attached to unactivated endothelium increased by 7 to 31 times after a 6-hour exposure of endothelium to tumor necrosis factor (TNF)-alpha. Inhibition studies showed that multiple adhesion receptors--including L-selectin, E-selectin, VCAM-1, and CD11/CD18--were involved in blast cell adhesion to TNF-alpha-activated endothelium. Leukemic cells were then cocultured at 37 degrees C on unactivated endothelial cell monolayers for time periods up to 24 hours. A time-dependent increase in the number of blasts attached to the endothelium and a concomitant induction of ICAM-1, VCAM-1, and E-selectin were observed. Additional experiments revealed that endothelial cell activation by leukemic myeloblasts was caused by cytokine secretion by blast cells, in particular TNF-alpha and IL-1 beta, and direct contacts between adhesion receptors expressed by blast cells and endothelial cells. Thus, leukemic cells have the ability to generate conditions that promote their own adhesion to vascular endothelium, a property that may have important implications for the pathophysiology of leukostasis and tissue infiltration by leukemic blast cells. (Blood. 2001;97:2121-2129)

Antigens, CD↗
PubMed2005

An in vitro approach to the chronological aging of skin by glycation of the collagen: the biological effect of glycation on the reconstructed skin model.

Glycation is a slow, nonenzymatic reaction that takes place between free amino groups in proteins primarily from lysine and a reducing sugar such as glucose or ribose. In skin, this reaction creates new residues or formations of cross-links (advanced glycation end products, AGEs) in the extracellular matrix of the dermis. The formation of these bridges between dermal molecules is supposed to be responsible for loss of elasticity or other properties of the dermis observed during aging. Glycation may therefore play an important role in chronologic aging. In order to examine this hypothesis, we have developed a reconstructed skin model made of a modified dermal compartment that is a fibroblast-contracted collagen lattice prepared with preglycated collagen. The presence of AGEs (glycoxidation products) in the skin equivalents was evidenced using specific antibodies against carboxymethyllysine (CML). Several changes were observed after collagen glycation: (1) fibroblast shape and distribution (vimentin staining) were modified; (2) extracellular matrix molecules and the dermal-epidermal junction zone seemed to be enhanced (procollagen I and III, collagen IV and VII stainings); (3) stainings for beta1 and alpha6 integrins were also increased in the epidermal cell layer; and (4) collagenase activity was increased. To verify the biological effect of glycation, we used the well-known glycation inhibitor aminoguanidine. After aminoguanidine treatment, we found a low CML amount and decreased distribution of markers previously overexpressed in glycated skin constructs. These in vitro findings were at least in part related to aging in vivo and demonstrate an actual effect of glycation in skin aging.

Animals↗
PubMed2000

Cell cycle arrest and differential gene expression in HT-29 cells exposed to an aqueous garlic extract.

Epidemiological data show an inverse correlation between garlic consumption and the risk for colon cancer. To examine this relationship, HT-29 human adenocarcinoma cells were cultured in the presence and absence of an aqueous garlic extract. Garlic treatment resulted in a fraction of cells detaching from the culture flasks. These cells remained viable. Flow cell cytometry showed that untreated cells exhibited a normal distribution among phases of the cell cycle, with 12% of cells at the G2/M boundary. Of the garlic-treated cells remaining attached to the flask, 27% were present at the G2/M boundary. Treated cells that detached from the flask were found almost exclusively (89%) at the G2/M boundary. RNA fingerprinting and microarray analysis showed that expression of the gene for menin was twice as high in control cells as in detached treated cells. In contrast, expression of genes for epidermal growth factor receptor and integrin-alpha6 was nearly twice as high in detached treated cells as in control cells. These changes in gene expression were consistent with an arrest of the cell cycle at the G2/M boundary. Garlic's arrest of the cell cycle in human adenocarcinoma cells may explain in part its anticarcinogenic properties.

Adenocarcinoma↗
PubMed1995

Integrin alpha 6 is a differentiation antigen of human granulosa cells.

We previously reported a new murine monoclonal antibody (mAb), OG-1, raised against human granulosa cells (GC) and showed that the OG-1 antigen is a cell surface differentiation-related molecule expressed on GC of follicles and on large luteal cells in the early to midluteal phase. In this study, we further characterized the OG-1 antigen. An immunohistochemical study showed that the OG-1 antigen was also expressed on endometrial glandular and stromal cells and on first trimester and term placental trophoblasts. The antigen purified from endometrium, chorionic villi, or placenta by immunoaffinity chromatography using the mAb OG-1 consisted of a 120-kilodalton (kDa) protein. Partial amino acid sequencing of the 120-kDa protein purified from placenta revealed that the 16 amino acids from the N-terminus were identical to those of the heavy chain of human integrin alpha 6, the molecular mass of which is reportedly 118-126 kDa. We examined the tissue distribution of integrins alpha 6, beta 1, and beta 4, because integrin alpha 6 forms heterodimers with integrins beta 1 or beta 4. Indirect immunofluorescence staining of various follicles and corpora lutea using two known antiintegrin alpha 6 monoclonal antibodies (450-30A1 and GoH3) showed that integrin alpha 6 was expressed on granulosa and large luteal cells in a profile similar to that of the OG-1 antigen. Integrin beta 1 was expressed on granulosa and large luteal cells, but integrin beta 4 was undetectable. Integrin beta 1 was also expressed on thecal and stromal cells. In term placentas, the OG-1 antigen and integrins alpha 6 and beta 4 were expressed on trophoblasts, whereas integrin beta 1 was expressed on villous capillary endothelia. These results indicate that the mAb OG-1 recognizes integrin alpha 6, and that integrin alpha 6 is a differentiation-related antigen of human GC, suggesting the involvement of integrins in human folliculogenesis and luteal formation.

Adult↗
PubMed2006

The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells.

We describe a novel murine progenitor cell population localised to a previously uncharacterised region between sebaceous glands and the hair follicle bulge, defined by its reactivity to the thymic epithelial progenitor cell marker MTS24. MTS24 labels a membrane-bound antigen present during the early stages of hair follicle development and in adult mice. MTS24 co-localises with expression of alpha6-integrin and keratin 14, indicating that these cells include basal keratinocytes. This novel population does not express the bulge-specific stem cell markers CD34 or keratin 15, and is infrequently BrdU label retaining. MTS24-positive and -negative keratinocyte populations were isolated by flow cytometry and assessed for colony-forming efficiency. MTS24-positive keratinocytes exhibited a two-fold increase in colony formation and colony size compared to MTS24-negative basal keratinocytes. In addition, both the MTS24-positive and CD34-positive subpopulations were capable of producing secondary colonies after serial passage of individual cell clones. Finally, gene expression profiles of MTS24 and CD34 subpopulations were compared. These results showed that the overall gene expression profile of MTS24-positive cells resembles the pattern previously reported in bulge stem cells. Taken together, these data suggest that the cell-surface marker MTS24 identifies a new reservoir of hair follicle keratinocytes with a proliferative capacity and gene expression profile suggestive of progenitor or stem cells.

Animals↗
PubMed2006

Epidermal and craniofacial defects in mice overexpressing Klf5 in the basal layer of the epidermis.

Krüppel-like factor5 (Klf5) is a zinc-finger transcription factor normally expressed in the skin. Here, we show that overexpression of Klf5 in the basal layer of the epidermis during embryogenesis affects epidermal development and disrupts epithelial-mesenchymal interactions necessary for skin adnexae formation as well as craniofacial morphogenesis. The transgenic mice exhibited exencephaly, craniofacial defects, persistent abdominal herniation and ectodermal dysplasia. Moreover, the epidermis was hypoplastic and underwent abnormal differentiation with expression of keratin8, a marker for single-layered epithelia, in the stratified epidermis. Correspondingly, we observed a downregulation of DeltaNp63 expression in the skin. Overexpression of Klf5 in adult mice led to hyperkeratosis, follicle occlusion and epidermal erosions. Further, we observed decrease and even loss of the stem cell population of bulge keratinocytes, as characterized by the expression pattern of alpha6 integrin and CD34 markers. Our data suggest a new role of Klf5 as a modulator of p63 expression and the differentiation program of epidermal cells relevant for regenerative potential of the epidermis and epithelial-mesenchymal interactions.

Animals↗
PubMed1994

Quantitative measurement of alpha 6 beta 1 and alpha 6 beta 4 integrin internalization under cross-linking conditions: a possible role for alpha 6 cytoplasmic domains.

In epithelial cells integrins are segregated on discrete domains of the plasma membrane. Redistribution may also occur during migration or differentiation. However, little is known about the mechanisms that control such redistribution. Receptor internalization may be a part of one such mechanism. We developed a quantitative assay and measured internalization of two epithelial integrin heterodimers, alpha 6 beta 1 and alpha 6 beta 4, induced by cross-linking with specific antibodies. alpha 6 beta 1 is a receptor for EHS laminin, while alpha 6 beta 4 is a receptor for a component of the basement membrane. alpha 6 beta 4 plays an important role in the establishment of hemidesmosomes, and becomes redistributed on the epithelial cell surface when cells are in a migratory phase. We report that alpha 6 beta 4 is efficiently internalized in human keratinocytes. More than 25% of cell surface alpha 6 beta 4 was internalized at 30 minutes, after cross-linking with A9, an anti-beta 4 monoclonal antibody. alpha 6 beta 1 is also internalized, in melanoma and teratocarcinoma cells, with maximum values of 20% of total receptors expressed at the cell surface. No significant difference was observed between the alpha 6 isoforms A and B in these assays. To determine whether alpha 6 cytoplasmic domains could influence integrin endocytosis, we prepared chimeric constructs with the extracellular domain of a reporter protein (CD8), and the cytoplasmic domains of either alpha 6 A or alpha 6 B. Both alpha 6 cytoplasmic domains but not a control cytoplasmic domain promoted internalization of the chimeric proteins, after cross-linking with antibody. Internalization of alpha 6 integrins may have a role in redistributing these receptors at the cell surface. Furthermore, the cytoplasmic domains of alpha 6 may be involved in regulating integrin internalization.

Animals↗
PubMed2000

Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by alpha 3 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrins.

Laminin-10/11, the laminin isoforms containing the alpha 5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin alpha 3 beta 1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin beta 1 antibody inhibited the adhesion of all cell types tested, anti-alpha 3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-alpha 3 and anti-alpha 6 antibodies, suggesting that both alpha 3 beta 1 and alpha 6 beta 1 integrins function as laminin-10/11 receptors in these cells. To explore this possibility, we examined the adhesion of K562 leukemic cells transfected with integrin alpha 3 or alpha 6 subunit to laminin-10/11 or other laminin isoforms. Laminin-10/11 were potent adhesive ligands for both the alpha 3 beta 11 and alpha 6 beta 1 transfectants, whereas laminin-5 was the preferred ligand for the alpha 3 beta 1 transfectants. Upon stimulation with the activating anti-integrin beta 1 antibody, both transfectants became more adherent to the substratum regardless of the type of laminins coated, although their preference for laminin isoforms remained unaltered. K562 cells transfected with alpha 6 and beta 4 subunits were also capable of adhering to laminin-10/11, indicating that integrin alpha 6 beta 4 is another receptor for laminin-10/11. Even with lung carcinoma cells, the alpha 6-containing integrins partly contributed to adhesion to laminin-10/11 at higher coating concentrations, although non-integrin receptor(s) might also be involved under such conditions. These results indicated that laminin-10/11 are potent and versatile adhesive ligands in basement membranes capable of binding to both alpha 3 beta 1 and alpha 6 beta 1 integrins with high avidity and also to alpha 6 beta 4 integrin.

Antigens, CD↗