An-Najah Staff

ABSTRACT Heart failure is considered as a major health problem worldwide featured by fibrosis & dysregulation of the extracellular matrix (ECM) homeostasis. Cardiac fibroblasts (CF) play a major role in fibrogenesis; as they are responsible for the production of ECM as well as the secretion of some important profibrotic mediators such as CTGF. Very little is known about the mechanisms controlling the production and secretion of ECM and profibrotic mediators by CF. Therefore, we intend to study the role of the cytoskeleton in fibrotic processes in CF, because cytoskeletal proteins such as tubulin and actin do not only provide physical support to cells, but also they play a vital role in the regulation of some cellular processes such as protein expression and secretion. Two different concentrations of the microtubule disrupting drug colchicine (1.3µg/mL & 5µg/mL) were studied in neonatal rat CF (NRCF). In the same time, the effect of angiotensin-II, a known physiological inducer for CTGF expression and secretion, was also studied in these cells. We found that both colchicine concentrations significantly reduced tubulin levels (by approximately ≈ 70%) and at the same time significantly increased CTGF expression (about 3 fold) & secretion (about 4 fold) , these effects were interestingly not associated with increased collagen synthesis. In the same context, the effect of angiotensin-II on colchicine-treated cells was almost abolished. Co-fluorescent staining of tubulin and actin in colchicine-treated cells showed that actin filaments were still intact and normally organised, in the time that microtubules were almost totally disrupted, also in another experiment where we selectively disrupted actin filaments by latrunculin A, there was no increase in the basal level of CTGF expression and secretion indicating that CTGF induction by colchicine might exclusively come from tubulin disruption . Moreover Immunofluorescent staining of CTGF in untreated cells clearly showed that CTGF granules are normally confined to the perinuclear microdomain comparing to colchicine-treated cells, where CTGF granules were diffuse within the cytosol, which further stresses on a vital role for tubulin on the regulation of CTGF handling in NRCF. Furthermore to see if the effect of colchicine in NRCF is RhoA-Rho kinase (ROCK) mediated as already demonstrated before in immortalized renal fibroblasts by Ott et al, 2003 & Graness et al, 2006, the ROCK inhibitor H1152P was tested along with colchicine in NRCF. We found that ROCK inhibition had no effect on colchicine-induced CTGF expression and secretion. In parallel , we studied the role of RhoA in CTGF handling by knocking down RhoA in NRCF using Sh-RhoA. These cells exhibited disorganized actin filaments, clear morphological changes and significant increase in tubulin level (≈ 25%) which was surprisingly associated with a significant increase in CTGF secretion and a statistically insignificant increase in CTGF expression. On one hand, this data provide an evidence that RhoA is involved in the modulation of CTGF handling and the homeostasis of tubulin, but on the other hand it suggest that RhoA activation might not be involved in the mechanism of CTGF induction by colchicine. In conclusion, our data suggest a prominent role for tubulin in the regulation of CTGF handling in a way that does not interfere with collagen synthesis , and is independent on RhoA-ROCK signalling.