Ceramide & capacity for growth in aging: a liver aspect

这项研究的长期目标是破译衰老过程中肝脏有丝分裂反应降低的机制。在完整的肝脏中，DNA合成的诱导需要最初从分化模式切换到增殖模式。这种能力是通过在不同的损伤模型中细胞-细胞接触的中断而实现的，例如肝部分切除或胶原酶灌流。获能是生长反应的关键；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，神经酰胺是一种参与生长停滞和分化的生物活性分子：胶原酶诱导的肝生物基质消化使肝细胞获得能力，与神经鞘磷脂酶活性下调及其产物神经酰胺水平下降有关。这些变化导致即时早期癌基因的诱导，这些癌基因是肝细胞获能的标志。相反，肝细胞在基质上的种植类似于天然的肝脏ECM，与正常的神经酰胺水平和对生长因子的反应能力有关。这些研究的主要目标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对以下假设进行批判性检验：(1)衰老涉及鞘磷脂信号转导通路的结构性上调，导致损伤诱导的肝细胞获能减弱；(2)鞘脂信号下调导致肝细胞增殖状态。在第一个特定目标中，我们将通过(I)比较年轻和老年动物在胶原酶处理期间立即早期癌基因的诱导性和下调神经鞘磷脂酶活性的能力，以及(Ii)在体内测试在胶原酶灌流期间保持高神经酰胺水平是否会阻止肝细胞的获能，来探讨老年动物中鞘磷脂酶的结构性激活是否干扰了肝细胞的正常获能。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细胞外基质对肝细胞生长能力的影响。首先，我们将测试ECM是否调节肝细胞中神经酰胺的水平。此外，我们将研究细胞神经酰胺的增加是否抑制生长能力，以及这些变化是否下调c-myc的表达。这些实验将评估神经酰胺和鞘磷脂酶在肝细胞获能中的作用，并将为进一步研究神经酰胺介导细胞外基质对肝细胞增殖能力的影响的机制提供实验基础。

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.