Figure 1.

TGFβ induces hyaluronan synthesis in NMuMG cells via upregulation of HAS2 mRNA in a p38- and Smad-dependent manner. NMuMG cells were starved and stimulated for 24 h (a and b) or 6 h (c) with TGFβ, in the absence or presence of TβRI-kinase inhibitor GW6604, p38 MAPK inhibitor SB203580 or dimethyl sulfoxide control, or the hyaluronan degrading enzyme Streptomyces hyaluronidase. Cells were pretreated with the above agents or enzyme for 1 h before stimulation. (a) Conditioned medium was collected and subjected to an enzyme-linked immunosorbent-like assay for analysis of hyaluronan amount. The average of three separate experiments performed in triplicates ±s.d. is shown. (b) Before starvation and stimulation, cells were transfected with scrambled siRNA or siRNA against Smad4. Following 24 h of stimulation, the hyaluronan amount was determined. A representative experiment out of three is shown ±s.d. (c) Total RNA was prepared and reversely transcribed to complementary DNA and real-time PCR was run with primers for HAS1, 2 and 3. The mRNA copy number was related to that of 18S ribosomal RNA housekeeping gene and the average of three separate experiments ±s.d. is shown. (d) Before starvation and TGFβ stimulation, cells were transfected with scrambled siRNA or siRNA against HAS2. Following 24 h of stimulation, the amount of hyaluronan was determined. A representative experiment out of three is shown ±s.d. P-values were calculated using Student's paired t-test and *P<0.05 were considered statistically significant compared with scramble. Asterisks indicate statistically significant differences compared with cells stimulated with TGFβ alone or as indicated with lines.