Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell-cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.H yaluronan (HA) is a linear polysaccharide composed of a, and is widely present as a major component of extracellular matrix in most vertebrate tissues (1, 2). HA is synthesized by three HA synthase isoforms: HAS1, HAS2, and HAS3 (3, 4). The three HAS genes have distinct expression patterns during mouse development (5), and their products have significantly different enzymatic properties and roles in the formation of the HA matrix (6).HA synthesis and matrix formation may have a role in the development, progression and pathogenesis of cancer. For example, the rate of HA synthesis is enormously increased when oncogenic viruses transform fibroblasts (7,8), and elevated levels of HA are associated with the hyperproliferative and malignant phenotypes in melanomas and various carcinomas (9-11).Studies done with cultured cancer cells showed that overproduction of HA enhances their anchorage-independent growth, tumorigenicity, and metastatic potential (12, 13), suggesting an important role for HA in tumor growth and malignant progression. However, it was unclear whether the overproduction could induce a malignant transformation of nontransformed cells.We overexpressed the three HAS isoforms to test the ability of HA to transform nontransformed cells. We also investigated whether the three HAS isoforms are functionally distinct in nontransformed cells. Materials and MethodsCell Culture and Transfection. 3Y1 1-B6 cells were obtained from Riken Cell Bank (Tsukuba, Japan). The 3Y1 cells were cultured in DMEM containing 10% FCS and 2 mM L-glutamine (growth medium). Stable transfectants were established as previously described (6) and were routinely cultured in ...

Malignant transformation of fibroblasts and epithelial cells is often accompanied by increased hyaluronan production and accumulation. Despite recent progress in the study of hyaluronan biosynthesis, the mechanisms underlying the transformation-induced overproduction of hyaluronan have not been elucidated. Here we report that activity and transcriptional levels of hyaluronan synthase (HAS) significantly increased after oncogenic malignant transformation of a rat 3Y1 fibroblast cell line. Of three HAS isoforms (HAS1, HAS2, and HAS3), only HAS2 gene expression was increased in the v-Ha-ras transformed 3Y1 cells, which show less malignancy. In contrast, both HAS1 and HAS2 expressions were elevated in the highly malignant cells transformed with v-src and/or v-fos. To assess the contribution of HAS expression to the oncogenic malignant transformation, we established stable cell transfectants expressing sense and antisense HAS genes. Antisense suppression of the HAS2 expression significantly decreased hyaluronan production in the cells transformed by the oncogenic v-Ha-ras and eventually led to a reduction in tumorigenicity in the rat peritoneum. The introduction of the HAS1 and HAS2 genes promoted the growth of subcutaneous tumors in a manner dependent on the levels of hyaluronan synthesis. Significant growth promotion was observed within a wide range of HAS1 expression. In contrast, the growth stimulation was only seen within a narrow range of HAS2 expression, and high levels of HAS2 expression even inhibited tumor growth. These results suggest that proper regulation of the expression of each HAS isoform is required for optimal malignant transformation and tumor growth.

We have used the yeast one-hybrid system to clone transcription factors that bind to specific sequences in the proximal promoters of the type I collagen genes. We utilized as bait the sequence between ؊180 and ؊136 in the pro-␣2(I) collagen promoter because it acts as a functional promoter element and binds several DNA-binding proteins. Three cDNA clones were isolated that encoded portions of the mouse SPR2 transcription factor, whereas a fourth cDNA contained a potential open reading frame for a polypeptide of 775 amino acids and was designated BFCOL1. Recombinant BFCOL1 was shown to bind to the ؊180 to ؊152 segment of the mouse pro-␣2(I) collagen proximal promoter and to two discrete sites in the proximal promoter of the mouse pro-␣1(I) gene. The N-terminal portion of BFCOL1 contains its DNA-binding domain. DNA transfection experiments using fusion polypeptides with the yeast GAL4 DNA-binding segment indicated that the C-terminal part of BFCOL1 contained a potential transcriptional activation domain. We speculate that BFCOL1 participates in the transcriptional control of the two type I collagen genes.Type I collagen is a protein that is abundantly synthesized by a discrete number of cell types including osteoblasts, odontoblasts, fibroblasts, smooth muscle cells, and mesenchymal cells. It is composed of two ␣1 chains and one ␣2 chain forming a characteristic triple helix. Expression of the genes for these polypeptides is coordinately regulated in a variety of physiological and pathological situations (1). Changes in the synthesis of type I collagen occur not only during embryonic development in specific tissues but changes also take place in disease states, for example during wound healing as well as in fibrotic diseases such as lung fibrosis, cirrhosis, and scleroderma. In many of these instances it is likely that the control of expression of the two type I collagen genes is mainly exerted at the level of transcription, but the precise mechanisms that control transcription of these genes are still poorly understood. Our long term goal is to identify the critical cis-acting elements in these two genes and both the cell-specific and ubiquitous transcription factors that presumably control their expression.Recently, transgenic mouse studies have identified strong tissue-specific enhancer elements in the 5Ј-flanking regions of both type I collagen genes (2-5). These elements are located further upstream than the proximal promoter elements. For instance, in the mouse pro-␣1(I) gene, a potent enhancer element for osteoblast and odontoblast expression was localized about 1.6 kilobases (kb) 1 upstream of the start of transcription, whereas another strong element for expression in tendon and fascia fibroblasts was found between Ϫ2.3 and Ϫ3.2 kb (2). Similar experiments from other laboratories have produced analogous results (3, 4). These experiments strongly suggested that separate elements control the expression of this gene in different type I collagen-producing cells. In the pro-␣2(I) gene, an element that strongly...

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148(+/-) mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148(+/-) tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation. Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp148(+/-) embryonic stem cells and in fetal germ cells from chimeric Zfp148(+/-) embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.