Intracellular hyaluronan protects murine fibroblasts against apoptosis (1003.3)

Abstract

Skin repair typically involves apoptotic cell death. We are interested in the role of hyaluronan (HA), an extracellular matrix molecule, during fibroblast‐mediated repair of injured skin. Has1/3 null mice (Has1/3), which lack hyaluronan synthases 1 and 3 (yet retain Has2) show accelerated wound closure, more fibroblasts, and fewer cleaved caspase‐3–positive dermal cells in healing wounds, suggestive of reduced apoptosis. In cultured Has1/3 fibroblasts, HA levels and Has2 mRNA expression were higher than in WT cells. Apoptosis, induced by serum starvation (SS) or UVB exposure, was reduced in the Has1/3 cells as shown by DNA fragmentation and by cleavage of caspases and PARP. Hyaluronidase, added to remove extracellular HA, surprisingly showed no effect on apoptotic susceptibility to SS or UVB. However, cells treated with 4‐MU to inhibit HA synthesis were sensitized to apoptosis induced by SS or UVB. To further establish a role for HA in protection from apoptosis, Has2 RNA interference (RNAi) was done using Has2‐specific siRNA transfection. Has2 RNAi in WT and Has1/3 fibroblasts inhibited HA synthesis by 90% and significantly increased their sensitivity to apoptosis. External addition of high MW HA failed to reverse this effect. We conclude that Has1/3 skin fibroblasts (which make relatively more HA) are more resistant to apoptosis due to the presence of intracellular HA synthesized by Has2.Grant Funding Source: supported by NHLBI