Retarded intracellular lipid transport associated with reduced expression of Cdc42, a member of Rho-GTPases, in human aged skin fibroblasts: A possible function of Cdc42 in mediating intracellular lipid transport

Objective - Many cell types in atherosclerotic lesions are thought to have various biological abnormalities, such as impaired lipid homeostasis and slow cell proliferation, which may be related to senescence at cellular and individual levels. One of the common characteristics of senescent cells in vitro is the alteration of actin cytoskeletons, which have been reported to be involved in the intracellular transport of lipids. Recently, we raised the hypothesis that Cdc42, which is a member of the Rho-GTPase family and is known to play an important role in actin dynamics, might be important in cellular lipid transport. Methods and Results - In the present study, we found that the protein expression levels and GTP-binding activities of Cdc42 were decreased in aged human skin fibroblasts. Moreover, we found the intracellular kinetics of Golgi-associated lipids to be retarded in these cells, which was demonstrated by the fluorescence recovery after photobleaching (FRAP) technique and the use of N-[7-(4-nitrobenzo-2-oxa-1,3- diazole)]-6-aminohexanoyl-D-erythro-sphingosine as a tracer. To correlate the decreased expression of Cdc42 with the retarded FRAP, we complemented the amount of wild-type c-myc-tagged Cdc42Hs (myc-Cdc42Hs-WT) by adenovirus-mediated gene transfer. We further tested the effect of the dominant-active form (myc-Cdc42Hs-DA, V12Cdc42Hs) or dominant-negative form (myc-Cdc42Hs-DN, N17Cdc42Hs) of Cdc42Hs on FRAP. Introduction of myc-Cdc42Hs-WT or myc-Cdc42Hs-DA recovered the retarded FRAP in the aged fibroblasts. Conversely, control fibroblasts infected with myc-Cdc42Hs-DN exhibited significantly retarded FRAP. Conclusions - These data clearly indicate that the expression of Cdc42, a small G protein, is decreased in the aged cells in close association with the retarded intracellular lipid transport. The present study demonstrates a possible function of Cdc42 in the mediation of intracellular lipid transport.