Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration - PubMed (original) (raw)
Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration
Naoki Itano et al. Proc Natl Acad Sci U S A. 2002.
Abstract
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.
Figures
Figure 1
Effects of HAS overexpression on cell density and morphology of confluent rat 3Y1 cells. (A) Rat 3Y1 fibroblasts were transfected with control vector or with HAS expression vectors, and clonal transfectants were established (6). HA coat formations (arrows) were detected by the particle exclusion assay. The parental cells were transfected with each HAS cDNA, and the G418-resistant transfectants were pooled. The transfectants were plated in 10-cm tissue culture dishes at 5 × 105 cells/dish and cultured for 14 days. The pooled HAS transfectants showed a spindle-like morphology and overlapping cell layers. (B) Western blotting analyses of membrane fractions isolated from the transfectants expressing FLAG-tagged HAS protein. Arrowheads indicate protein bands of the FLAG-tagged HAS1 (68 kDa), HAS2 (60 kDa), and HAS3 proteins (60 kDa). (C) HA synthase activities of confluent cultures were determined as described in Materials and Methods. The HA contents of the conditioned medium of exponentially growing and confluent cultures were measured by a competitive ELISA-like assay as described in Materials and Methods. Column, mean values of three determinations; bars, SD.
Figure 2
Morphological and cytoskeletal alterations of confluent HAS2 transfectants. Confluent cultures of control transfectants (A, C, E, and G) and HAS2 transfectants (B, D, F, and H) were analyzed by scanning electron microscope (A and B) and by staining with rhodamine-phalloidin (C and D), pan anti-cadherin antibody (E and F) or biotinylated HA binding protein (HABP; G and H). Scanning electron micrographs revealed a number of filopodial protrusions at the cell edges of the HAS2 transfectants (B). The confluent HAS2 transfectants changed their actin filament organization from cortical actin filaments to actin stress fibers (D). The distribution of cadherin at the intercellular boundaries was significantly altered in the HAS2 transfectants (F). Bars = 5 μm (A and B) and 20 μm (C_–_H).
Figure 3
Cell cycle progression of contact-inhibited 3Y1 cells by HA overproduction. Cell cycle profiles were monitored by flow cytometry at 24 h after the pulsing of confluent cell cultures with BrdUrd. Cells labeled with BrdUrd were prepared as described in the Materials and Methods. All HAS transfectants had a high proportion of cells in S phase and G2/M phase, and a low percentage in G0/G1 phase, as compared with the control cells.
Figure 4
Inhibition of the PI3-kinase pathway resulted in reacquisition of normal phenotype of the HAS2 transfectants. The HAS2 stable transfectants were cultured with (B, D, F, H, and J) or without (A, C, E, G, and I) 10 μM of LY294002, a PI3-kinase inhibitor, just after the cells reached confluence, and cultured for additional 5 days in the presence of the inhibitor. The HAS2 transfectants treated with the inhibitor showed normal morphology (B). Rhodamine-phalloidin staining of actin showed reappearance of pericellular actin rings in the HAS2 transfectants treated with the inhibitor (D). Immunofluorescent staining of N-cadherin and β-catenin in HAS2 transfectants demonstrated that PI3-kinase inhibition resulted in the accumulations of N-cadherin and β-catenin at the cell borders (F and H) whereas HA matrix was maintained on the HAS2 transfectants even after the treatment with LY294002 (J). Wortmannin (50 μM), a potent PI3-kinase inhibitor, also gave similar effects on the phenotypic changes of the HAS2 transfectants (data not shown).
References
- Laurent T C, Fraser J R E. FASEB J. 1992;6:2397–2404. - PubMed
- Knudson C B, Knudson W. FASEB J. 1993;7:1233–1241. - PubMed
- Weigel P H, Hascall V, Tammi M. J Biol Chem. 1997;272:13997–14000. - PubMed
- Itano N, Kimata K. Trend Glycosci Glycotechnol. 1998;10:23–38.
- Spicer A P, McDonald J A. J Biol Chem. 1998;273:1923–1932. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials