Laminin-binding integrins are essential for the maintenance of functional secretory epithelium in lactation [luminal cells]
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ABSTRACT: Integrin dimers α3/β1, α6/β1 and α6/β4 are the mammary epithelial cell receptors for laminins, which are major components of the basement membrane, a specialized extracellular matrix surrounding the mammary epithelium. 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 by the Cre-Lox approach. During pregnancy, mutant mice displayed low levels of luminal progenitor activity and retarded lobulo-alveolar development, whereas their mammary glands seemed to be functional at the onset of lactation. Myoepithelial cell morphology was markedly altered in mutant glands, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. However, lactation was not sustained in mutant mice, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. This study reveals an essential role for laminin-binding integrins in functional mammary gland development.
Project description:Integrin dimers α3/β1, α6/β1 and α6/β4 are the mammary epithelial cell receptors for laminins, which are major components of the basement membrane, a specialized extracellular matrix surrounding the mammary epithelium. 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 by the Cre-Lox approach. During pregnancy, mutant mice displayed low levels of luminal progenitor activity and retarded lobulo-alveolar development, whereas their mammary glands seemed to be functional at the onset of lactation. Myoepithelial cell morphology was markedly altered in mutant glands, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. However, lactation was not sustained in mutant mice, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. This study reveals an essential role for laminin-binding integrins in functional mammary gland development.
Project description:A goal of this project is to evaluate the integrin mRNA expression in human neural stem/progenitor cells (hNSPC) using high-throughput sequencing technologies. We found high levels of mRNA expression for the β1, α7, α3, α6, β5, αV, α5, and α9 integrins. This suggests that hNSPCs may express integrin receptors that can bind fibrinogen and laminin proteins.
Project description:Integrin dimers a3/b1, a6/b1 and a6/b4 are the receptors of mammary epithelial cells for Laminin, major component of the mammary basement membrane. Antibodies against a6 and a3 integrin chains serve to isolate stem cell enriched populations from mammary epithelium, however, role of integrin dimers comprising these chains in the control of mammary stem cell activity are not known. To investigate the role of Laminin-binding integrins in the control of mammary stem cell function, a3 and a6 chains were deleted from mammary basal cells in vitro using Cre-recombinase carrying adenovirus. We found that deletion of single integrin chain (either a3, or a6) did not significantly affect stem cell potential as evaluated by transplantation and mammosphere assays, whereas basal cells depleted of both a3 and a6 integrin chains (a3a6KO) presented severely diminished stem cell activity. In this study the transcriptional profiles of a3a6KO were analysed and compared to those of control cells.
Project description:Analysis of gene expression with cDNA array showed high expression levels of apoptosis activator Apaf1, membrane proteins CD44 and TGFR1β, integrin family members α2, α3, α6, αV and β1, p53 regulator MDM2, p21Ras GTP-ase activating molecule RasA1, and extracellular matrix-associated protein thrombospondin I. Expression of EGFR confirmed the immunocytochemical results, and expression of integrin α6 and CD44 support basal epithelial character of the EM-G3 cells. Keywords: cDNA array, breast cancer cell line
Project description:Gene-level and exon-level analysis of gene expression in MDA-MB-231 cells that stably express control shRNA or integrin α3-targeting shRNA. The laminin-332-binding integrin α3b1 is expressed highly in many breast cancer cells, but its roles in regulating gene expression programs that promote breast cancer progression have not been explored. In order to identify genes that are regulated by α3b1 in human breast cancer cells, we used a lentiviral approach to express an α3-targeting shRNA to suppress integrin α3b1 in MDA-MB-231 cells, and we identified subsequent changes in gene expression and alternate exon useage. We used the Affymetrix Human Exon 1.0 ST platform to analyze biological replicates of MDA-MB-231 cells that were transduced with lentivirus to stably express either control shRNA or α3-targeting shRNA. Array data was processed by Affymetrix Exon Array Computational Tool.
Project description:Gene-level and exon-level analysis of gene expression in MDA-MB-231 cells that stably express control shRNA or integrin α3-targeting shRNA. The laminin-332-binding integrin α3b1 is expressed highly in many breast cancer cells, but its roles in regulating gene expression programs that promote breast cancer progression have not been explored. In order to identify genes that are regulated by α3b1 in human breast cancer cells, we used a lentiviral approach to express an α3-targeting shRNA to suppress integrin α3b1 in MDA-MB-231 cells, and we identified subsequent changes in gene expression and alternate exon useage.
Project description:Integrins are among the most abundant cell surface receptors constituting the principal adhesion receptors for the extracellular matrix (ECM), providing a physical anchor for the cell and triggering multiple intracellular signalling events. Loss-of function mutations of the integrin α3 gene (ITGA3) have been recently disclosed in patients with interstitial lung disease, congenital nephrotic syndrome and junctional epidermolysis bullosa, a multiorgan disorder with fatal outcome. In these patients, the respiratory function is strongly impaired and the kidneys are variably affected, whereas skin fragility is rather mild, has a delayed onset after birth or remains unrecognized, suggesting that integrin α3 differently influences the development and homeostasis of these organs. Here we employed authentic human keratinocytes bearing a naturally occurring integrin α3 loss-of-function mutation as a prototype to characterize the molecular mechanisms launched by the constitutional absence of this integrin subunit. To validate our findings, we generated new cellular models, including an additional ILNEB patient cell line, ITGA3 rescued and knockdown cells. We show that keratinocytes lacking a functional α3 subunit have an activated cellular phenotype with a switch in the pattern of integrin α subunits on the cell surface. These assure spreading and adhesion of epidermal keratinocytes but also drive the migratory phenotype of these cells.
Project description:Mammary epithelium is composed by luminal and basal epithelial cells, which are adhere to the basement membrane (BM). To dissect how basal cell functions are regulated by BM laminin adhesion, we performed RNA sequencing of basal human mammary epithelial cells adhered on laminin-111, -421 or -521 coated cell culture plates for 48 hours.
Project description:The molecular mechanisms that regulate breast cancer cell (BCC) metastasis and proliferation within the leptomeninges (LM) are poorly understood, limiting development of effective therapies. Here we show that BCCs in mice can invade the LM by abluminal migration along blood vessels that connect vertebral/calvarial bone marrow and meninges, bypassing the blood-brain barrier. This process is dependent on BCC engagement with vascular basement membrane laminin through expression of the neuronal pathfinding molecule integrin α6. Once in the LM, BCCs co-localize with perivascular meningeal macrophages and induce their expression of the pro-survival neurotrophin, GDNF. Intrathecal GDNF blockade, macrophage-specific GDNF ablation, or deletion of the GDNF receptor, NCAM, from BCCs, inhibit BC growth within LM. Here, we performed RNA-seq analysis of GDNF-treated and/or anti-NCAM1-treated EO771-tdT-LM2 cells under glucose-deprived media treatment.
Project description:Previously we have shown significant differences in lactation performance, mammary gland histology and expression profiles of mammary transcriptome during peak-lactation (lactation day 9; L9) between the ordinary CBA/CaH (CBA) and the superior QSi5 strains of mice. In the present study, we compared mammary gland histology between CBA and QSi5 at mid-pregnancy (pregnancy day 12; P12). We assessed lactation performance during the first 8 days of lactation of the 13th - 14th generation of the Advanced Intercross Line (AIL) (CBA X QSi5) mice. We utilized an integrative approach to analyzing mammary microarray expression profiles of CBA and QSi5 at P12 and CBA, AIL and QSi5 at L9. The inguinal mammary glands of CBA/CaH and QSi5 during mid-pregnancy (Pregnancy day 12; P12), and the glands of CBA/CaH, AIL and QSi5 during peak lactation (Lactation day 9; L9) were collected and total RNA was extracted for Affymetrix microarray (mouse genome 430 2) assay