Intercellular communication controls agonist-induced calcium oscillations independently of gap junctions in smooth muscle cells.

In this study, we report the existence of a communication system among human smooth muscle cells that uses mechanical forces to frequency modulate long-range calcium waves. An important consequence of this mechanical signaling is that changes in stiffness of the underlying extracellular matrix can interfere with the frequency modulation of Ca2+ waves, causing smooth muscle cells from healthy human donors to falsely perceive a much higher agonist dose than they actually received. This aberrant sensing of contractile agonist dose on stiffer matrices is completely absent in isolated smooth muscle cells, although the isolated cells can sense matrix rigidity. We show that the intercellular communication that enables this collective Ca2+ response in smooth muscle cells does not involve transport across gap junctions or extracellular diffusion of signaling molecules. Instead, our data support a collective model in which mechanical signaling among smooth muscle cells regulates their response to contractile agonists.

Roles for beta(pat-3) integrins in development and function of Caenorhabditis elegans muscles and gonads.

Heterodimeric integrin receptors for extracellular matrix (ECM) play vital roles in bidirectional signaling during tissue development, organization, remodeling, and repair. The beta integrin subunit cytoplasmic domain is essential for transmission of many of these signals and overexpression of an unpaired beta tail in cultured cells inhibits endogenous integrins. Unlike vertebrates, which have at least nine beta subunit genes, the nematode Caenorhabditis elegans expresses only one beta subunit (betapat-3), and a null mutation in this gene causes embryonic lethality. To determine the functions of integrins during larval development and in adult tissues, we have taken a dominant negative approach by expression of an HA-betatail transgene composed of a hemagglutinin (HA) epitope tag extracellular domain connected to the betapat-3 transmembrane and cytoplasmic domains. Expression of this transgene in muscle and gonad, major sites of integrin expression, caused a variety of phenotypes dependent on the level of transgene expression. Abnormalities in body wall and sex muscles led to uncoordinated movement and egg-laying defects. Significant anomalies in migration and pathfinding were caused by tissue-specific expression of HA-betatail in the distal tip cells (DTC), the cells that direct gonad morphogenesis. A pat-3 gene with Tyr to Phe mutations in the cytoplasmic domain was able to rescue pat-3 null animals but also showed DTC migration defects. These results show that betapat-3 plays important roles in post-embryonic organogenesis and tissue function.

Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter.

Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetables, can induce a G(1) cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analysis of CDK6 mRNA decay rates revealed that I3C had no effect on CDK6 transcript stability. We report the first identification and functional characterization of the CDK6 promoter in order to determine whether I3C inhibits CDK6 transcription. In MCF-7 cells stably transfected with CDK6 promoter-linked luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C-specific response that was not a consequence of the growth-arrested state of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between -805 and -638. Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are spaced 5 base pairs apart, were necessary for I3C responsiveness in the context of the CDK6 promoter. Electrophoretic mobility shift analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells, in combination with supershift assays using Sp1 antibodies, demonstrated that the Sp1-binding site in the CDK6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 transcription factor. Taken together, our results suggest that I3C down-regulates CDK6 transcription by targeting Sp1 at a composite DNA site in the CDK6 promoter.

Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells.

The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The naturally occurring chemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone. This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage-independent growth, reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2-specific enzymatic activity than either compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs, whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.

Glucocorticoids stimulate p21 gene expression by targeting multiple transcriptional elements within a steroid responsive region of the p21waf1/cip1 promoter in rat hepatoma cells.

Glucocorticoids can induce a G1 arrest in the cell cycle progression of BDS1 rat hepatoma cells. In these cells, dexamethasone, a synthetic glucocorticoid, stimulated a rapid and selective increase in expression of the p21 cyclin-dependent kinase (CDK) inhibitor mRNA and protein and virtually abolished CDK2 phosphorylation of the retinoblastoma protein. Expression of the p27 CDK inhibitor, and other G1-acting cell cycle proteins, remained unaffected. Dexamethasone stimulated p21 promoter activity in a p53-independent manner that required functional glucocorticoid receptors. Transforming growth factor-beta, which also induced a G1 cell cycle arrest of the hepatoma cells, failed to elicit this response. Analysis of 5' deletions of the p21 promoter uncovered a glucocorticoid responsive region between nucleotides -1481 and -1184, which does not contain a canonical glucocorticoid response element but which can confer dexamethasone responsiveness to a heterologous promoter. Fine mapping of this region uncovered three distinct 50-60-base pair transcriptional elements that likely function as targets of glucocorticoid receptor signaling. Finally, ectopic expression of p21 had no effect on hepatoma cell growth in the absence of glucocorticoids but facilitated the ability of dexamethasone to inhibit cell proliferation. Thus, our results have established a direct transcriptional link between glucocorticoid receptor signaling and the regulated promoter activity of a CDK inhibitor gene that is involved in the cell cycle arrest of hepatoma cells.

Role of the CCAAT/enhancer binding protein-alpha transcription factor in the glucocorticoid stimulation of p21waf1/cip1 gene promoter activity in growth-arrested rat hepatoma cells.

The preceding paper (Cha, H. H., Cram, E. J., Wang, E. C., Huang, A. J., Kasler, H. G., and Firestone, G. L. (1998) J. Biol. Chem. 273, 0000-0000(478563) defined a glucocorticoid responsive region within teh promoter of the p21 CDK inhibitor gene that contains a putative DNA-binding site for the transcription factor CCAAT/ enhancer binding protein-alpha (C/EBP alpha). Wild type rat BDS1 hepatoma cells as well as as4 hepatoma cells, which express antisense sequences to C/EBP alpha and ablate its protein production, were utilized to investigate the role of this transcription factor in the glucocorticoid regulation of p21 gene expression. The stimulation of p21 protein levels and promoter activity, as well as inhibition of CDK2-mediated retinoblastoma protein phosphorylation, by the synthetic glucocorticoid, dexamethasone, required the expression of C/EBP alpha. Overexpression of C/EBP alpha in as4 cells rescued the dexamethasone responsiveness of the p21 promoter. Site-directed mutagenesis of the p21 promoter revealed that dexamethasone stimulation of p21 promoter activity required the C/EBP consensus DNA-binding site. Furthermore, in glucocorticoid receptor-defective EDR1 hepatoma cells, dexamethasone failed to stimulate C/EBP alpha and p21 protein expression and promoter activities. Our results have established a functional link between the glucocorticoid receptor signaling pathway that mediates a G1 cell cycle arrest of rat hepatoma cells and the transcriptional control of p21 by a cascade that requires the steroid induction of C/EBP alpha gene expression.