Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI
- PDF / 2,491,047 Bytes
- 17 Pages / 595.276 x 790.866 pts Page_size
- 55 Downloads / 141 Views
ORIGINAL PAPER
Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI3K-AKT and p38 signaling Sanna Pasonen-Seppa¨nen • Piia Takabe • Michael Edward • Leena Rauhala • Kirsi Rilla Markku Tammi • Raija Tammi
•
Accepted: 6 July 2012 / Published online: 24 July 2012 Ó Springer-Verlag 2012
Abstract In many cancers hyaluronan content is increased, either by tumor cells or the surrounding stromal cells and this increased hyaluronan content correlates with unfavorable clinical prognosis. In the present work, we studied the effects of melanoma cell (aggressive melanoma cell line C8161)-derived factors on fibroblast hyaluronan synthesis, intracellular signaling, MMP expression and invasion. Treatment of the fibroblast cultures with melanoma cell conditioned medium (CM) caused accumulation of hyaluronan in the culture medium and formation of thick pericellular hyaluronan coat and hyaluronan cables. The expression of Has2 was increased approximately 20-fold by the C8161 melanoma cell CM, while Has1 and Has3 were increased twofold. Knock-down of Has2 expression with siRNA showed that Has2 was responsible for the increased hyaluronan synthesis induced by the melanoma cell CM. To find out the signaling routes, which led to Has2 upregulation, the phosphorylation profiles of 46 kinases were screened with phosphokinase array kit. Melanoma cell CM treatment strongly induced a rapid phosphorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and cJUN. Treatment of the fibroblasts with specific inhibitors of PI3K, AKT and p38 reduced the melanoma cell CM-
induced hyaluronan secretion, while the inhibitor of PDGFR totally blocked it. In addition, siRNA for PDGFRa/b inhibited Has2 upregulation in melanoma cell CM-treated fibroblasts. In parallel with the increased hyaluronan synthesis the melanoma cell CM-treated fibroblasts showed spindle shape, numerous long cell protrusions, enhanced MMP expression and increased invasion into collagen-Cultrex matrix. siRNA blocking of Has2 or PDGFRa/b expression reversed the stimulatory effect of melanoma cell CM on fibroblast invasion. PDGF secreted by melanoma cells thus mediated fibroblasts activation, with HAS2 upregulation as a major factor in the fibroblast response. This effect on stromal matrix is suggested to favor tumor growth. Keywords Hyaluronan Melanoma Fibroblast Interaction Invasion Extracellular matrix Abbreviations CM Conditioned medium ECM Extracellular matrix bHABC Biotinylated hyaluronan binding complex ELSA Enzyme-linked sorbent assay HAS Hyaluronan synthase
Electronic supplementary material The online version of this article (doi:10.1007/s00418-012-1000-x) contains supplementary material, which is available to authorized users. S. Pasonen-Seppa¨nen (&) P. Takabe L. Rauhala K. Rilla M. Tammi R. Tammi School of Medicine, Institute of Biomedicine/Anatomy, University of Eastern Finland, Kuopio, Finland e-mail: [email protected] M. Edward Section of Dermatology, School of Medicine, University of Glas
Data Loading...
