TGF-beta1 regulates the expression of multiple max-interacting transcription factors in Balb/MK cells: implications for understanding the mechanism of action of TGF-beta1.

Appropriate transforming growth factor-beta1 (TGF-beta1) signaling is required to preserve homeostasis of diverse tissues during development. At the cellular level, one function of TGF-beta1 that is critical for preserving homeostasis is the ability to arrest cell growth. TGF-beta1 arrests growth by blocking the function of the c-myc proto-oncogene. c-myc function is determined by the level of c-myc expression relative to other Max-interacting transcription factors, and TGF-beta1 has been shown to inhibit c-myc expression by inhibiting c-myc transcription. However, whether TGF-beta1 might also increase the expression of a Max-interacting factor that blocks myc function by competing with myc for Max binding is not known. Therefore, we determined the effect of TGF-beta1 on the expression of Max-interacting transcription factors in Balb/MK cells. We found unexpectedly that Balb/MK cells express both N-myc and c-myc. The pattern of N-myc expression during the cell cycle differs from that of c-myc, indicating that mRNA accumulation is controlled by mechanisms specific to each gene. TGF-beta1 rapidly inhibits N-myc mRNA expression; thus N-myc is a novel target of TGF-beta1 in Balb/MK cells. More importantly, we found that TGF-beta1 induces the expression of the putative tumor suppressor genes Mad4 and Mxi1 in both the Balb/MK and Mv1Lu cell lines. Mad4 and Mxi1 are novel targets of TGF-beta1, known to inhibit cell growth by antagonizing the interaction of Myc with Max. Thus, our results suggest that the induction of Mad4 and Mxi1 may function in tandem with the inhibition of N-myc and c-myc to mediate the growth inhibitory function of TGF-beta1.

Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism.

Transforming growth factor-beta1 (TGF-beta) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-beta are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-beta-induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-beta do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-beta rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160(ROCK), by the expression of dominant-negative mutants, inhibited TGF-beta-mediated EMT. The data suggest that TGF-beta rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.

Salicylate-induced growth arrest is associated with inhibition of p70s6k and down-regulation of c-myc, cyclin D1, cyclin A, and proliferating cell nuclear antigen.

Salicylate and its pro-drug form aspirin are widely used medicinally for their analgesic and anti-inflammatory properties, and more recently for their ability to protect against colon cancer and cardiovascular disease. Despite the wide use of salicylate, the mechanisms underlying its biological activities are largely unknown. Recent reports suggest that salicylate may produce some of its effects by modulating the activities of protein kinases. Since we have previously shown that the farnesyltransferase inhibitor l-744, 832 inhibits cell proliferation and p70(s6k) activity, and salicylate inhibits cell proliferation, we examined whether salicylate affects p70(s6k) activity. We find that salicylate potently inhibits p70(s6k) activation and phosphorylation in a p38 MAPK-independent manner. Interestingly, low salicylate concentrations (/=5 mm) are required to block p70(s6k) activation by epidermal growth factor + insulin-like growth factor-1. These data suggest that salicylate may selectively inhibit p70(s6k) activation in response to specific stimuli. Inhibition of p70(s6k) by salicylate occurs within 5 min, is independent of the phosphatidylinositol 3-kinase pathway, and is associated with dephosphorylation of p70(s6k) on its major rapamycin-sensitive site, Thr(389). A rapamycin-resistant mutant of p70(s6k) is resistant to salicylate-induced Thr(389) dephosphorylation.

Overexpression of the c-Myc oncoprotein blocks the growth-inhibitory response but is required for the mitogenic effects of transforming growth factor beta 1.

One of the more intriguing aspects of transforming growth factor beta 1 (TGF beta 1) is its ability to function as both a mitogenic factor for certain mesenchymal cells and a potent growth inhibitor of lymphoid, endothelial, and epithelial cells. Data are presented indicating that c-myc may play a pivotal role in both the mitogenic and antiproliferative actions of TGF beta 1. In agreement with previous studies using C3H/10T1/2 fibroblasts constitutively expressing an exogenous c-myc cDNA, we show that AKR-2B fibroblasts expressing a chimeric estrogen-inducible form of c-myc (mycER) are able to form colonies in soft agar in the presence of TGF beta 1 only when c-myc is activated by hormone. Whereas these findings support a synergistic role for c-myc in mitogenic responses to TGF beta 1, we also find that c-myc can antagonize the growth-inhibitory response to TGF beta 1. Mouse keratinocytes (BALB/MK), which are normally growth-arrested by TGF beta 1, are rendered insensitive to the growth-inhibitory effects of TGF beta 1 upon mycER activation. This ability of mycER activation to block TGF beta 1-induced growth arrest was found to occur only when the fusion protein was induced with hormone in the early part of G1. Addition of estradiol late in G1 had no suppressive effect on TGF beta 1-induced growth inhibition.

Inhibition of cell growth by TGF beta 1 is associated with inhibition of B-myb and cyclin A in both BALB/MK and Mv1Lu cells.

The concept of positive and negative regulation of normal cellular growth by diffusible factors is well illustrated by the effects of epidermal growth factor and transforming growth factor beta 1 (TGF beta 1) on mouse keratinocytes (MK) and mink lung epithelial cells (Mv1Lu). MK and Mv1Lu are nontransformed cell lines that reversibly arrest at a point in late G1 in response to TGF beta 1. Previously, we have shown that expression of the protooncogene c-myc is induced upon epidermal growth factor stimulation of quiescent MK and Mv1Lu cells and that transcriptional suppression of c-myc by TGF beta 1 treatment is important in the TGF beta 1 growth inhibition pathway. Using epidermal growth factor-stimulated synchronized MK and Mv1Lu cells, we have investigated the mRNA expression of a large number of growth factor-inducible genes that are critical regulators of growth in G1 and at the G1/S transition. These genes, often found to be dysregulated in cancer, include transcription factors as well as cyclins and their associated kinases, that promote growth, and tumor suppressor genes, that inhibit growth. As reported here, TGF beta 1 significantly inhibited mRNA expression of B-myb and cyclin A in both cell lines, suggesting that these may be important common downstream targets in the growth inhibition pathway. In contrast, the expression patterns of cyclins D1 and D2 and the transcription factors E2F1 and E2F2 were unaffected in MK cells treated with TGF beta 1 but were significantly inhibited in TGF beta 1-treated Mv1Lu cells. We cite the evidence suggesting that the inhibition of B-myb and cyclin A may contribute to the late G1 arrest caused by TGF beta 1 and that these events may be linked through the actions of the product of the retinoblastoma susceptibility gene (Rb) or an Rb family member.