Identification of the LIM kinase-1 as a ceramide-regulated gene in renal mesangial cells

Stimulation of rat renal mesangial cells with cell-permeable C6-ceramide for 6 and 24 h induces the expression of several genes as analyzed by a RNA fingerprinting arbitrarily primed-PCR method. Sequencing of the differentially expressed bands identified the serine/threonine protein kinase LIM kinase-1 (LIMK-1), which is involved in the regulation of cytoskeletal organization, as a ceramide-induced gene. The ceramide-triggered upregulation of LIMK-1 was verified by semiquantitative reverse transcriptase-PCR. A detailed time course reveals a first detectable increase in RNA level after 2 h of ceramide stimulation which reaches maximal levels after 6 It of stimulation and remains elevated up to 24 h. This ceramide-induced gene transcription of LIMK-I is accompanied by enhanced LIMK-I protein levels with maximal protein expression seen after 6 h of stimulation. Furthermore, cofilin, which is a specific substrate of LIMK-1, shows an increased phosphorylation at Ser-3 in mesangial cells exposed to C6-ceramide. Mechanistically, the ceramide-induced LIMK-I expression is blocked by the Rho kinase inhibitor Y27632, but not by a farnesyl transferase inhibitor, suggesting the involvement of the small G protein Rho, but not Ras and Rac, in the expressional upregulation. Similar to exogenously added ceramide, also interleukin-1beta which is an established activator of the neutral sphingomyelinase that leads to endogenous ceramide formation upregulates LIMK-1 protein expression and activity. In summary, these data demonstrate for the first time that LIMK-1 is a ceramide-induced gene, thus suggesting that LIMK-l may act as a link between stress-induced ceramide formation and reorganization of the actin cytoskeleton. (C) 2002 Elsevier Science (USA). All rights reserved.