Mad1 function is regulated through elements within the carboxy terminus.

Myc and Mad are basic helix-loop-helix leucine zipper (bHLH-LZ) proteins that heterodimerize with Max to bind DNA and thereby influence the transcription of Myc-responsive genes. Myc-Max dimers transactivate whereas Mad-Max-mSin3 complexes repress Myc-mediated transcriptional activation. We have previously shown that the N-terminal mSin3 binding domain and the centrally located bHLH-LZ are required for Mad1 to function during a molecular switch from proliferation to differentiation. Here we demonstrate that the carboxy terminus (CT) of Mad1 contains previously unidentified motifs necessary for the regulation of Mad1 function. We show that removal of the last 18 amino acids of Mad1 (region V) abolishes the growth-inhibitory function of the protein and the ability to reverse a Myc-imposed differentiation block. Moreover, deletion of region V results in a protein that binds DNA weakly and no longer represses Myc-dependent transcriptional activation. In contrast, deletion of the preceding 24 amino acids (region IV) together with region V restores DNA binding and transcriptional repression, suggesting a functional interplay between these two regions. Furthermore, phosphorylation within region IV appears to mediate this interplay. These findings indicate that novel regulatory elements are present in the Mad1 CT.

Thyroid hormone-induced cell proliferation in GC cells is mediated by changes in G1 cyclin/cyclin-dependent kinase levels and activity.

The thyroid hormone, 3,3', 5-triiodo-L-thyronine (T3), is essential for growth and regulation of metabolic functions. The biological activities of T3 are mediated by its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. We found that T3 stimulated cell growth in GC cells by shortening the doubling time approximately 3-fold. Flow cytometric analysis indicated that the growth stimulatory effect was mainly due to shortening of G1 phase accompanied by increases in S and G2/M phases of the cell cycle. These changes correlated with T3-induced increases in messenger RNA and protein levels of two key regulators of G1 progression, cyclins D1 and E, as well as cdk2. Furthermore, the kinase activities associated with cyclin D1 and E were activated up to 4-fold by T3, which led to increased phosphorylation of the retinoblastoma protein (Rb), the driving force in G1 to S cell cycle progression. These results show for the first time that the growth promoting effect of T3 in GC cells is mediated, at least in part, by increases in cyclin/cdk activities and the phosphorylation state of Rb. The functional link of T3 to Rb has important implications for the understanding of the biology of normal and cancer cells.

Thyroid hormone receptor is a negative regulator in p53-mediated signaling pathways.

Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcription factors which regulate growth, differentiation, and development. The molecular mechanism by which TRs mediated these effects remains unclear. A prevailing hypothesis is that TRs exert their biological effects by cooperating with other transcription factors. We have recently shown that the human TR subtype beta1 (hTRbeta1) interacts with the tumor suppressor p53, which plays a critical role in cell-cycle regulation and tumorigenesis. This interaction of hTRbeta1 with p53 leads to an impairment of TR function. The present study examined whether hTRbeta1 could modulate the function of p53. Mapping of the domains of p53 responsible for the interaction with hTRbeta1 indicated that the regions involved resided in the DNA-binding domain and carboxy terminus of p53. In agreement with this finding, hTRbeta1 increased the binding of p53 to p53 DNA-binding elements. This increase in DNA binding, however, resulted in repression of p53-dependent transcription activation in transfected cells. Furthermore, hTRbeta1 led to an inhibition of the p53-mediated induction of bax and gadd45 expression. In contrast, the p53-induced expression of p21 was not affected by hTRbeta1, suggesting that the expression of p53-regulated genes is differentially modulated by hTRbeta1. Because the expressions of bax, gadd45, and p21 are directly regulated by p53, these results indicate that hTRbeta1 can modulate p53-regulated gene expression and support the hypothesis that there is cross-talk between these two regulatory pathways. The cross-talk between these two transcription factors could play an important role in the biology of normal and cancer cells.

The tumor suppressor p53 is a negative regulator of estrogen receptor signaling pathways.

The estrogen receptor (ER) is a ligand-dependent transcription factor which regulates growth, development, differentiation and reproduction. To test the hypothesis that the diverse effects of the ER could be mediated by interacting with other transcription factors/oncogenes, the present study assessed its interaction with the tumor suppressor p53. p53 is a transcription factor which is involved in cell cycle regulation and apoptosis. We found that the wild-type p53 physically interacted with ER in vivo and repressed the estrogen-activated transcriptional activity. However, p53 mutants had no or reduced repression effect, depending on the sites of mutation. These findings suggest that p53 can cross talk with the ER in hormone-activated signaling pathways in cells.

Phlorizin or vanadate treatment reverses impaired expression of albumin and hepatocyte nuclear factor 1 in diabetic rats.

Diabetes decreases transcription of the albumin gene. The role of hyperglycemia in mediating this suppression of albumin gene activity is unclear. To study the effect of glucose in vivo, we treated diabetic rats with phlorizin or vanadate, two agents that ameliorate hyperglycemia without increasing the levels of circulating insulin. When glucose was normalized in diabetic rats with either agent, the hepatic levels of albumin mRNA became indistinguishable from those in nondiabetic animals. In light of our previous observation that diabetes decreases the abundance of hepatocyte nuclear factor 1 (HNF1), the predominant factor increasing albumin gene transcription, we wondered whether glucose normalization in diabetes would alter HNF1. Both the levels and DNA binding activity of HNF1 were restored to control values when phlorizin or vanadate was administered to diabetic rats. These findings suggest that hyperglycemia is integrally involved in mediating the suppression of albumin gene expression in diabetes. The effect of hyperglycemia on HNF1 suggests that glucose affects albumin expression at the level of transcription.

Effects of diabetes mellitus on hepatocyte nuclear factor 1 decrease albumin gene transcription.

We have previously reported that albumin gene transcription is reduced in diabetes mellitus (DM). The present study explored the mechanism by which albumin gene transcription is down-regulated in DM. Deletional studies and displacement of factors binding to site B of the albumin promoter indicated that the repressive effects of DM are mediated by nuclear factors binding to this site. Since hepatocyte nuclear factor 1 (HNF1) activates albumin promoter activity and is the predominant factor binding to site B, we examined HNF1. The abundance and binding activity of HNF1 were reduced in hepatonuclear extracts from diabetic compared to control rats. However, HNF1 mRNA levels were unchanged, suggesting that the effect of DM on HNF1 is at the post-transcriptional level. Extracts from diabetic animals also contained another protein, distinct from HNF1 and vHNF1, which bound to site B in gel retardation studies. In summary, our studies demonstrate that the reduced abundance and binding activity of HNF1 correlates with decreased albumin gene transcription in DM.