Phospholipase C (PLC) links many extracellular signals to intracellular responses via the hydrolysis of phosphoinositide 4,5-biphosphate (PIP2). Although much is known about the regulation of the PLCβ and PLCλ isoforms, the mechanism of regulation of PLCδ1 remains obscure. PLCδ1 is the most predominant isoform in differentiated, contractile HVSMC and has been proposed to be involved in cytoskeletal organisation (Lymn & Hughes, 2000). Previous studies in our group have suggested that PLCδ1 activity is dually regulated by GTP proteins with Rho A acting as the negative modulator (Hodson et al. 1998). Tissue transglutaminase (TGII/Gh) is a dual-functional enzyme, which can act either as a transglutaminase involved in protein modification and cross-linking or as a GTPase. TGII/Gh has previously been demonstrated to play a role in PLCδ1 activation in rat liver (Feng et al. 1996).
The aim of this study is to determine the level of TGII/Gh expression and cellular localisation in relation to PLCδ1 expression and redifferentiation of HVSMC.
HVSMC, derived from saphenous vein by a routine explant technique, were cultured in Dulbecco’s modified Eagle’s medium containing 15 % fetal calf serum. The use of this tissue conformed to local ethics commitee guidelines. In order to induce a measure of redifferentiation HVSMC were serum deprived for 7 days. Expression of smooth muscle α-actin (SM α-actin), PLCδ1 and TGII/Gh were determined by SDS-PAGE and Western blotting. Cellular location was determined using confocal microscopy. Protein expression in randomly cycling cells was taken to be 100 % and all the data were expressed as means ± S.E.M. with respect to this. Statistical analysis was performed using Student’s paired t test and a P value < 0.05 was considered as significant. Serum deprivation of HVSMC resulted in a significant increase in α-actin, PLCδ1 and Rho A protein expression, while TGII/Gh expression was significantly decreased.
Serum deprivation resulted in α-actin becoming more structured with fibres running the length of the cell. PLCδ1 also took on a more structured appearance. Intriguingly, PLCδ1 and TGII/Gh were expressed in different locations in randomly cycling cells, but were co-localised in serum-deprived cells.
In conclusion, serum deprivation modulates both PLCδ1 and TGII/Gh protein expression and induces a cellular association of these enzymes. These data suggest that TGII/Gh may play a role in determining not only PLCδ1 but also contraction.
This work was supported by a British Heart Foundation grant awarded to J.S. Lymn and A.D. Hughes.
All procedures accord with current UK legislation.