Abstract
BackgroundConnective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells. An HC11 mammary epithelial cell line expressing CTGF/CCN2 was constructed to dissect the cellular responses to CTGF/CCN2 that contribute to this differentiation program.ResultsTetracycline-regulated expression of CTGF/CCN2 in HC11 cells enhanced multiple markers of lactogenic differentiation including β-casein transcription and mammosphere formation. In a separate measure of mammary differentiation the addition of CTGF/CCN2 to cultures of MCF10A cells increased the development of acini in vitro. In HC11 cells the elevated levels of CTGF/CCN2 diminished the requirement for extracellular matrix proteins in the activation of β-casein transcription, indicating that CTGF/CCN2 contributed to lactogenic differentiation through the regulation of matrix dependent cell adhesion. CTGF/CCN2 expression in HC11 cells increased expression of extracellular matrix proteins and integrins, enhanced the formation of focal adhesion complexes, and increased survival signaling. In addition, HC11 cells adhered to immobilized CTGF/CCN2 and this was inhibited by function-blocking antibodies to the integrins α6 and β1, and to a lesser degree by antibody to β3 integrin.ConclusionsCTGF/CCN2 expression in HC11 cells led to an increase in multiple markers of lactogenic differentiation. The mechanisms by which CTGF/CCN2 contributed to lactogenic differentiation include direct binding of CTGF/CCN2 to integrin complexes and CTGF/CCN2-induced matrix protein expression resulting in elevated integrin functionality.
Highlights
Connective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells
Because functional b1 integrin complexes are required for lactogenesis in vivo and in vitro, our studies focused on the effect of CTGF/CCN2 expression on this axis in mammary epithelial cells
Ectopic CTGF/CCN2 expression enhanced the lactogenic differentiation of HC11 cells Previous studies conducted in our laboratory demonstrated that CTGF/CCN2 was transcriptionally regulated by the glucorticoid dexamethasone during the induction of lactogenic differentiation in HC11 cells, and that elevated CTGF/CCN2 expression was required for the differentiation program [27]
Summary
Connective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells. Lactogenic differentiation is associated with the deposition of laminin-rich matrix by the epithelial cells [6,7] and the degree of differentiation of mammary epithelial cells correlates with their response to basement membrane and stromal protein-induced signals. B1 integrin expression is required for the survival of epithelial cells during differentiation [19] and it contributes to mammary gland development and morphogenesis [20,21]. In vitro studies of the interaction between mammary epithelial cells and basement membrane proteins during transcription of milk proteins recently implicated the SWI/SNF transcription factor, Brg, in translating signals from the stroma to the activation of the b-casein promoter [26]
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