Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in vitro. To assess HA functionality, HAS expression was inhibited in SW620 cells by transfection with antisense HAS constructs. Decreased HA secretion and retention in the transfectants were confirmed using competitive binding and particle exclusion assays. SW620 cells demonstrated greater invasion through Matrigel than did SW480 cells. Antisense transfection decreased Matrigel invasion by SW620 cells by >60%; addition of exogenous HA restored invasion. Because the cell-surface HA receptor CD44 has been implicated in cancer progression, HA-CD44 interaction was then inhibited by incubation with an anti-CD44 antibody. Anti-CD44 antibody impaired invasion into Matrigel by 95%. Taken together, these data suggest that pericellular HA is critical for colon carcinoma cell invasion and that this invasive capability is dependent on interaction with CD44.

HA is a glycosaminoglycan that is synthesized on the inner surface of the plasma membrane and secreted into the pericellular matrix. HA and its biosynthetic enzymes (HAS1, HAS2 and HAS3) are thought to participate in tumor growth and cancer progression. In our study, colon carcinoma cells isolated from a lymph node metastasis (SW620) produced more pericellular HA and expressed higher levels of HAS3 mRNA compared to cells isolated from a primary colon carcinoma (SW480). To assess functionality, HAS3 expression in SW620 cells was inhibited by transfection with an asHAS3 construct. Decreased HA secretion and cell-surface retention by asHAS3 transfectants were confirmed using competitive binding and particle exclusion assays. Anchorage-independent growth, a correlate of tumor growth in vivo, was assessed by colony formation in soft agar. SW620 cells stably transfected with asHAS3 demonstrated significant growth inhibition, as evidenced by fewer colonies and smaller colony area than either SW620 cells or cells transfected with vector alone. Addition of exogenous HA restored growth in asHAS3 transfectants. Thus, we demonstrate that pericellular HA secretion and retention and HAS3 expression are increased in metastatic colon carcinoma cells relative to cells derived from a primary tumor. Inhibition of HAS3 expression in these cells decreased the pericellular HA matrix and inhibited anchorage-independent growth. These data suggest that HA and HAS3 function in the growth and progression of colon carcinoma.

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Protein tyrosine phosphatases (PTPs) are critical regulators of cellular phosphorylation functioning in processes such as cell growth, differentiation, and adhesion. Osteotesticular PTP (OST) is the only characterized member of this superfamily whose expression is regulated in osteoblasts and critical for their in vitro differentiation. Such evidence would suggest that this molecule is a key modulator of signaling events during osteogenesis, yet little is known about its genetic regulation. In an effort to examine the molecular mechanisms involved in the cellular regulation of OST, we have characterized its expression in MC3T3 osteoblasts during differentiation. Northern analysis revealed that murine OST mRNA is dramatically regulated during the preosteoblast to osteoblast progression, with predominant expression in differentiated and early mineralizing osteoblasts. This expression pattern is unique to this phosphatase since, in comparison, the structurally similar receptor PTP, LAR, and the intracellular PTP1B show little change during differentiation. Cell density contributes to this upregulated expression as confluent cultures display an increase in OST transcripts within 4 h post-plating. Transient transfection of the OST promoter in differentiating MC3T3 results in a significant increase in transcriptional activation from day 0 to day 5 of differentiation, similar in timing and intensity to the observed upregulation of the endogenous gene. This activation appears to be specific to osteoblasts, since progression to a myoblast phenotype results in no change in reporter gene activity. Culturing these preosteoblast cells in the absence of critical co-factors results in an inhibition of differentiation and leads to a delayed induction of OST transcripts as well as the attenuation of transcriptional activation. These results show that the murine OST gene is regulated at the transcriptional level in an osteoblast-specific, differentiation-dependent manner during the differentiation of MC3T3 osteoblasts. Future studies will help determine the essential regulatory elements within the OST-PTP promoter and the critical signaling pathways important in this regulation.