Nitric oxide induces degradation of the neutral ceramidase in rat renal mesangial cells and is counterregulated by protein kinase C
Nitric oxide induces degradation of the neutral ceramidase in rat renal mesangial cells and is counterregulated by protein kinase C
复制标题
DOI：
10.1074/jbc.m204034200
复制
发表时间：
2002-11-29
影响因子：
4.8
通讯作者：
Huwiler, A
中科院分区：
文献类型：
--
作者：
Franzen, R;Fabbro, D;Huwiler, A
一氧化氮（NO）刺激肾系膜细胞可使神经酰胺水平显著升高。这种效应先前被证明是由于一氧化氮的双重作用，包括鞘磷脂酶的激活和神经酰胺酶活性的抑制。在这项研究中，我们发现no触发的中性神经酰胺酶活性的抑制与蛋白质水平的下调是平行的。刺激24小时后中性神经酰胺酶蛋白完全丧失。选择性蛋白酶体抑制剂lactacystin阻断no诱导的神经酰胺酶降解，而几种caspase抑制剂无效。此外，no诱导的降解被蛋白激酶C （PKC）激活剂，12- o - tetradecanoyiphorbol13 -acetate (TPA)，以及生理PKC激活剂血小板衍生生长因子- bb (PDGF)，血管紧张素11和ATP逆转，导致中性神经酰胺酶蛋白及其活性正常化。使用P-32(i)标记的系膜细胞的体内磷酸化研究表明，TPA、PDGF、血管紧张素H和ATP触发中性神经酰胺酶的磷酸化增加，该磷酸化被广谱PKC抑制剂Ro-31 8220阻断，但不被CGP 41251阻断，CGP 41251对Ca2+依赖的异构体有优先作用，因此表明参与Ca2+独立的PKC异构体。利用未经刺激的系膜细胞免疫沉淀的重组PKC同工酶和中性神经酰胺酶进行的体外磷酸化实验表明，PKC- δ同工酶和PKC- α同工酶在较小程度上能有效地直接磷酸化中性神经酰胺酶。综上所述，我们的数据表明NO能够诱导中性神经酰胺酶的降解，从而促进神经酰胺在细胞中的积累。这种作用被PKC激活逆转，最有可能是PKC- α同工酶，它可以直接磷酸化，从而防止中性神经酰胺酶降解。这些新的调节相互作用将为靶向中性神经酰胺酶稳定性和随后的神经酰胺积累提供治疗上有价值的信息。
Ceramide levels are strongly increased by stimulation of renal mesangial cells with nitric oxide (NO). This effect was shown previously to be due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of ceramidase activity. In this study we show that the NO-triggered inhibition of neutral ceramidase activity is paralleled by a down-regulation at the protein level. A complete loss of neutral ceramidase protein is obtained after 24 h of stimulation. Whereas the selective proteasome inhibitor lactacystin blocked NO-evoked ceramidase degradation, several caspase inhibitors were ineffective. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-O-tetradecanoyIphorbol-13-acetate (TPA), and also by the physiological PKC activators platelet-derived growth factor-BB (PDGF), angiotensin 11 and ATP, resulting in a normalization of neutral ceramidase protein as well as activity. In vivo phosphorylation studies using P-32(i)-labeled mesangial cells revealed that TPA, PDGF, angiotensin H, and ATP trigger an increased phosphorylation of the neutral ceramidase, which is blocked by the broad spectrum PKC inhibitor Ro-31 8220 but not by CGP 41251, which has a preferential action on Ca2+-dependent isoforms, thus suggesting the involvement of a Ca2+-independent PKC isoform. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral ceramidase immunoprecipitated from unstimulated mesangial cells show that particularly the PKC-delta isoform and to a lesser extent the PKC-alpha isoform are efficient in directly phosphorylating neutral ceramidase. In summary, our data show that NO is able to induce degradation of neutral ceramidase, thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-alpha isoenzyme, which can directly phosphorylate and thereby prevent neutral ceramidase degradation. These novel regulatory interactions will provide therapeutically valuable information to target neutral ceramidase stability and subsequent ceramide accumulation.