Selective Expression and Functional Characteristics of Three Mammalian Hyaluronan Synthases in Oncogenic Malignant Transformation

Shun'Ichiro Taniguchi,Naoki Itano,Reiji Kannagi,Osamu Miyaishi,Fukiko Atsumi,Koji Kimata,Michinari Hamaguchi,Takahiro Sawai

doi:10.1074/jbc.m313178200

Abstract

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.

Highlights

Malignant transformation of fibroblasts and epithelial cells is often accompanied by increased hyaluronan production and accumulation
Both HAS1 and HAS2 expressions were elevated in the highly malignant cells transformed with v-src and/or v-fos
A good correlation was found between the hyaluronan synthase (HAS) activity and the HA production (Fig. 2), suggesting that the HA production enhanced by oncogenic transformation was mainly due to the up-regulation of the HAS activity

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—Rat 3Y1 fibroblast cell lines transformed by various oncogenes, E1A-3Y1, SV-3Y1, HR-3Y1, and SR-3Y1, were obtained from Riken Bioresource Center (Ibaraki, Japan). 3Y1, E1A-3Y1, SV-3Y1, HR-3Y1, SR-3Y1, and v-fos SR-3Y1 cell lines were cultured for 2 days, and the conditioned medium was recovered. The conditioned medium was recovered from S17N SR-3Y1 cells incubated with or without 2 ␮M dexamethasone (Sigma) for 2 days. Quantitative Analyses of the HAS Transcripts—Relative levels of HAS expression in oncogenic transformed cells were determined by real-time quantitative RT-PCR as described previously [23]. Establishment of Stable Transfectants Expressing HAS Genes—The cDNAs for mouse HAS1, HAS2, and HAS3 were subcloned into the pEXneo mammalian expression vector as described previously [22]. Estimation of the Amounts of FLAG-tagged Recombinant HAS Proteins—Amounts of FLAG-tagged HAS fusion proteins in HAS transfectants were estimated by immunoblotting as described previously [22]. Tumor Formation—The HR-3Y1 cells stably expressing sense or antisense HAS were cultured in the growth medium and harvested by trypsinization.

RESULTS

98 Ϯ 15 128 Ϯ 12

DISCUSSION