Ceramide & capacity for growth in aging: a liver aspect

本研究的长期目标是破译衰老过程中肝脏有丝分裂反应下降的机制。在完整的肝脏中，DNA合成的诱导需要从分化模式到增殖模式的初始转换。这种能化是通过在不同的损伤模型中破坏细胞间的接触来实现的，如部分肝切除术或胶原酶灌注。容能对生长反应至关重要；然而，目前只知道这一过程的几个基本方面。肝细胞的增殖能力与神经酰胺水平呈负相关，神经酰胺是一种生物活性分子，参与生长停滞和分化的开始：肝细胞通过胶原酶诱导的肝生物基质消化而获得能力，与鞘磷脂酶活性的下调及其产物神经酰胺水平的降低相关。这些变化继续诱导直接早期癌基因，这是肝细胞能化的标志。相反，将肝细胞移植到类似天然肝外基质上，神经酰胺水平正常且不能对生长因子产生反应。这些研究的主要目的是检验这些观察到的关系是否真的是因果关系。我们将使用体内和体外两种方法来严格检验以下假设：(1)衰老涉及鞘脂信号转导途径的组成性上调，导致损伤诱导的肝细胞容量的衰减；(2)鞘脂信号下调导致肝细胞增殖状态。在第一个具体目标中，我们将通过(I)比较年轻和年老动物在胶原酶治疗期间立即早期癌基因的诱导性和下调鞘磷脂酶活性的能力，以及（ii）在体内测试在胶原酶灌注期间维持高神经酰胺水平是否会阻断肝细胞的能化，来探索老年动物鞘磷脂酶的组成性激活是否会干扰肝细胞的适当能化。在第二个特定目标中，我们将在体外测试神经酰胺是否介导ECM对肝细胞生长能力的影响。首先，我们将测试ECM是否调节肝细胞中的神经酰胺水平。此外，我们将研究细胞神经酰胺的增加是否会抑制生长能力，以及这些变化是否会下调c-myc的表达。本实验将进一步探讨神经酰胺和鞘磷脂酶在肝细胞获能中的作用，为进一步研究神经酰胺介导ECM对肝细胞增殖能力影响的机制提供实验依据。

The long-term goal of this study is to decipher the mechanism for decreased mitogenic response of liver during aging. In intact liver the induction of DNA synthesis requires initial switch from differentiated to proliferative mode. This capacitation is achieved by disruption of cell- cell contacts during different injury models, such as partial hepatectomy or collagenase perfusion. Capacitation is critical for growth response; however only a few fundamental aspects of this process are known now. Proliferative capacity of hepatocytes inversely correlates with the level of ceramide, a bioactive molecule that is involved in the onset of growth arrest and differentiation: Capacitation of hepatocytes by collagenase- induced digestion of hepatic biomatrix correlates with down-regulation of sphingomyelinase activity and decrease in the level of its product, ceramide. These changes proceed the induction of immediate early oncogenes that are hallmarks of hepatocyte capacitation. Conversely, plating of hepatocytes on matrix similar to the natural hepatic ECM, is associated with normal levels of ceramide and inability to respond to growth factors. The major goal of these studies is to test whether these observed relationships are truly causal. We will use both, in vivo and to vitro methods to test critically the hypothesis that (1) aging involves constitutive up-regulation of sphingolipid signal transduction pathway, leading to attenuation of injury-induced hepatocyte capacitation; and (2) down-regulation of sphingolipid signaling causes proliferative state of hepatocytes. In the first specific aim we will explore whether constitutive activation of sphingomyelinase in old animals interferes with the proper capacitation of hepatocytes by (I) comparing the inducibility of immediate early oncogenes and ability to downregulate sphingomyelinase activity during the collagenase treatment in young and old animals, and (ii) testing in vivo whether maintaining ceramide level high during the collagenase perfusion blocks the capacitation of hepatocytes. In the second specific aim we will test in vitro whether ceramide mediates the effects of ECM on hepatocytes capacity to grow. First, we will test whether ECM regulates ceramide level in hepatocytes. Further, we will investigate whether increases in cellular ceramide inhibit ability to grow and whether these changes down-regulate c-myc expression. These experiments will evaluate the role of ceramide and sphingomyelinase in hepatocytes capacitation and will provide the experimental grounds for future studies on the mechanism by which ceramide mediates the effects of ECM on ability of hepatocytes to undergo proliferation.