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Abstract Background Connective 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. Results Tetracycline-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. Conclusions CTGF/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.

Abstract Background HC11 mouse mammary epithelial cells differentiate in response to lactogenic hormone resulting in expression of milk proteins including β-casein. Previous studies have shown that epidermal growth factor (EGF) blocks differentiation not only through activation of the Ras/Mek/Erk pathway but also implicated phosphatidylinositol-3-kinase (PI-3-kinase) signaling. The current study analyzes the mechanism of the PI-3-kinase pathway in an EGF-induced block of HC11 lactogenic differentiation. Results HC11 and HC11-luci cells, which contain luciferase gene under the control of a β-casein promotor, were treated with specific chemical inhibitors of signal transduction pathways or transiently infected/transfected with vectors encoding dominant negative-Akt (DN-Akt) or conditionally active-Akt (CA-Akt). The expression of CA-Akt inhibited lactogenic differentiation of HC11 cells, and the infection with DN-Akt adenovirus enhanced β-casein transcription and rescued β-casein promotor-regulated luciferase activity in the presence of EGF. Treatment of cells with Rapamycin, an inhibitor of mTOR, blocked the effects of EGF on β-casein promotor driven luciferase activity as effectively as PI-3-kinase inhibitors. While expression of CA-Akt caused a constitutive activation of p70S6 kinase (p70S6K) in HC11 cells, the inhibition of either PI-3-kinase or mTOR abolished the activation of p70S6K by EGF. The activation of p70S6K by insulin or EGF resulted in the phosphorylation of ribosomal protein S6 (RPS6), elongation initiation factor 4E (elF4E) and 4E binding protein1 (4E-BP1). But lower levels of PI-3-K and mTOR inhibitors were required to block insulin-induced phosphorylation of RPS6 than EGF-induced phosphorylation, and insulin-induced phosphorylation of elF4E and 4E-BP1 was not completely mTOR dependent suggesting some diversity of signaling for EGF and insulin. In HC11 cells undergoing lactogenic differentiation the phosphorylation of p70S6K completely diminished by 12 hours, and this was partly attributable to dexamethasone, a component of lactogenic hormone mix. However, p70S6K phosphorylation persisted in the presence of lactogenic hormone and EGF, but the activation could be blocked by a PI-3-kinase inhibitor. Conclusion PI-3-kinase signaling contributes to the EGF block of lactogenic differentiation via Akt and p70S6K. The EGF-induced activation of PI-3-kinase-Akt-mTOR regulates phosphorylation of molecules including ribosomal protein S6, eIF4E and 4E-BP1 that influence translational control in HC11 cells undergoing lactogenic differentiation.

CCCTC-binding factor (CTCF) is a highly conserved, ubiquitously expressed zinc finger protein. CTCF is a multifunctional protein, associated with a number of vital cellular processes such as transcriptional activation, repression, insulation, imprinting and genome organization. Emerging evidence indicates that CTCF is also involved in DNA damage response. In this review, we focus on the newly identified role of CTCF in facilitating DNA double-strand break repair. Due to the large number of cellular processes in which CTCF is involved, factors that functionally affect CTCF could have serious implications on genomic stability. It is becoming increasingly clear that exposure to environmental toxicants could have adverse effects on CTCF functions. Here we discuss the various ways that environmental toxicants could impact CTCF functions and the potential consequences on DNA damage response. [ABSTRACT FROM AUTHOR]

Oxygen levels range from 2% to 9% in vivo. Atmospheric O2 levels (21%) are known to induce cell proliferation defects and cellular senescence in primary cell cultures. However, the mechanistic basis of the deleterious effects of higher O2 levels is not fully understood. On the other hand, immortalized cells including cancer cell lines, which evade cellular senescence are normally cultured at 21% O2 and the effects of higher O2 on these cells are understudied. Here, we addressed this problem by culturing immortalized human bronchial epithelial (BEAS-2B) cells at ambient atmospheric, 21% O2 and lower, 10% O2. Our results show increased inflammatory response at 21% O2 but not at 10% O2. We found higher RelA binding at the NF-κB1/RelA target gene promoters as well as upregulation of several pro-inflammatory cytokines in cells cultured at 21% O2. RelA knockdown prevented the upregulation of the pro-inflammatory cytokines at 21% O2, suggesting NF-κB1/RelA as a major mediator of inflammatory response in cells cultured at 21% O2. Interestingly, unlike the 21% O2 cultured cells, exposure of 10% O2 cultured cells to H2O2 did not elicit inflammatory response, suggesting increased ability to tolerate oxidative stress in cells cultured at lower O2 levels. J. Cell. Physiol. 231: 1611-1620, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: Connective 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. Results: Tetracycline-regulated expression of CTGF/CCN2 in HC11 cells enhanced multiple markers of lactogenic differentiation including b-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 b-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 functionblocking antibodies to the integrins a6 and b1, and to a lesser degree by antibody to b3 integrin. Conclusions: CTGF/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. [ABSTRACT FROM AUTHOR]

The response of mammary epithelial cells to basement membrane and stroma induced signals contributes to the degree of differentiation in this tissue. The studies reported here indicate that connective tissue growth factor (CTGF) is highly elevated during lactogenic differentiation of the HC11 mouse mammary epithelial cell line. In addition, CTGF is expressed in the mouse mammary gland during pregnancy and lactation and it is expressed in primary mammary epithelial cell cultures established from pregnant mice. In HC11 cells CTGF is transcriptionally regulated by dexamethasone, but not by estrogen or progesterone, and CTGF expression is not dependent on TGFβ. CTGF contributes to and is required for lactogenic differentiation of HC11 cells, as demonstrated by increased differentiation following expression of plasmid-encoded CTGF and decreased differentiation following depletion of endogenous CTGF with siRNA. Moreover, HC11 mouse mammary epithelial cells infected with an adenoviral vector encoding CTGF exhibit increased lactogenic differentiation. Plasmid vector-induced elevation of CTGF levels also increased the level of β1 integrin in HC11 cells. Because the production of stromal factors is an important component of differentiation in mammary epithelial cells, the regulation of CTGF by glucocorticoids may play a critical role in this aspect of the control of differentiation. The studies reported here provide important information on the role of CTGF in mammary epithelial cell differentiation. J. Cell. Physiol. 214:38–46, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The response of mammary epithelial cells to basement membrane and stroma induced signals contributes to the degree of differentiation in this tissue. The studies reported here indicate that connective tissue growth factor (CTGF) is highly elevated during lactogenic differentiation of the HC11 mouse mammary epithelial cell line. In addition, CTGF is expressed in the mouse mammary gland during pregnancy and lactation and it is expressed in primary mammary epithelial cell cultures established from pregnant mice. In HC11 cells CTGF is transcriptionally regulated by dexamethasone, but not by estrogen or progesterone, and CTGF expression is not dependent on TGFbeta. CTGF contributes to and is required for lactogenic differentiation of HC11 cells, as demonstrated by increased differentiation following expression of plasmid-encoded CTGF and decreased differentiation following depletion of endogenous CTGF with siRNA. Moreover, HC11 mouse mammary epithelial cells infected with an adenoviral vector encoding CTGF exhibit increased lactogenic differentiation. Plasmid vector-induced elevation of CTGF levels also increased the level of beta1 integrin in HC11 cells. Because the production of stromal factors is an important component of differentiation in mammary epithelial cells, the regulation of CTGF by glucocorticoids may play a critical role in this aspect of the control of differentiation. The studies reported here provide important information on the role of CTGF in mammary epithelial cell differentiation., ((c) 2007 Wiley-Liss, Inc.)