In line with earlier studies on other cell types,
13–16,23,24 we have found that 4-MU in the same concentration range has inhibited HA synthesis in normal and GO OFs, as well as DFs examined, even in the presence of TGF-β. HA production of the OFs originated either from GO or non-GO control orbits decreased in the same extent as it was observed in the fibroblasts with dermal origin. This inhibition during 24-hour 4-MU treatment is mainly due to the well-known mechanism that 4-MU depletes the cellular pool of the HA synthesis precursor, UDP-GlcUA.
17 Because it is known that 4-MU treatment reduces the expression of HAS enzymes,
18 we measured the effect of 4-MU on the transcriptional levels of several enzymes involved in HA metabolism. We found here that 4-MU greatly reduced
HAS2 mRNA expression, whereas it increased mildly the
HAS3 expression in OFs and DFs. The same effect on
HAS2 expression was described in cancer cells in which the level of
HAS3 mRNA was also reduced except for 1 cell line with very low
HAS3 expression level.
18 We assume that the effect of 4-MU on transcription of HAS genes with very low expression levels is less consistent;
HAS2 expression was 2 orders of magnitude higher than
HAS1 and
HAS3 in the studied cells, as we described earlier.
11 This theory is supported by our observation that significant downregulating effect of 4-MU on the expression of
HAS1 was observed in TGF-β stimulated cells where
HAS1 transcription is substantially higher (
Fig. 6B). The direct effect of 4-MU on the expression of
HAS genes is still unclear, but it was described earlier that
HAS1 and
HAS2 expressions positively correlated with the cellular level of both UDP-GlcUA and UDP–N-acetyl-glucosamine,
25 suggesting a strong indirect effect of 4-MU due to the UDP-GlcUA depletion. No correlation was found between
HAS3 expression and intracellular UDP-sugar contents
25 suggesting that the effect of 4-MU on
HAS3 expression is less predictable. The contribution of the 4-MU induced changes in the expression pattern of HAS enzymes to the reduced HA production is uncertain given the fact that UDP-GlcUA starvation prevented the localization of the intact HAS on the plasma membrane,
26 where HA synthesis can occur.