Spontaneous apoptosis in primary cultures of human and rat hepatocytes: molecular mechanisms and regulation by dexamethasone

Abstract

To elucidate the biochemical pathways leading to spontaneous apoptosis in primary cultures of human and rat hepatocytes, we examined the activation of the caspase cascade, the expression of Bcl-2-related-proteins and heat shock proteins. Comparisons were made before and after dexamethasone (DEX) treatment. We show that DEX inhibited spontaneous apoptosis in a dose-dependent manner. DEX increases the expression of anti-apoptotic Bcl-2 and Bcl-x L proteins, decreases the expression of pro-apoptotic Bax and inhibits Bad translocation thereby preventing the release of cytochrome c, the activation of caspases, and cell death. Although, the expression of Hsp27 and Hsp70 proteins remained unchanged, the oncogenic protein c-Myc is upregulated upon DEX-treatment. These results indicate that DEX mediates its survival effect against spontaneous apoptosis by acting upstream of the mitochondrial changes. Thus, the mitochondrial apoptotic pathway plays a major role in regulating spontaneous apoptosis in these cells. Blocking this pathway therefore may assist with organ preservation for transplant, drug screening, and other purposes. Cell Death and Differentiation (2002) 9, 945 ± 955.

Dexamethasone protects primary cultured hepatocytes from death receptor-mediated apoptosis by upregulation of cFLIP

“…14 It has been also shown that DEX suppresses spontaneous apoptosis in primary cultured hepatocytes. 11 To investigate its antiapoptotic signaling mechanism in TNF-a þ ActD-induced hepatocyte apoptosis, we first established the optimal concentration for the inhibition of apoptosis by pretreatment with DEX for 6 h. When incubated with cells in the presence of TNF-a þ ActD, DEX pretreatment suppressed apoptotic cell death in a dosedependent manner as determined by morphologic changes ( Figure 1A), DNA fragmentation ( Figure 1B), and crystal violet staining ( Figure 1C). The maximal inhibition was observed at 5 mM of DEX.…”

“…9,10 The antiapoptotic effect by DEX is regulated by several sets of genes, the best characterized of which are the Bcl-2 family such as Bcl-2 and Bcl-xL, which prevent spontaneous apoptosis of cultured primary hepatocytes. 11 It is also known that DEX abolishes TNF-a-induced cytoxicity of several tumor cell lines via inhibition of phospholipase A 2 activity. 12,13 However, the mechanism behind the antiapoptotic effects of DEX has rarely been investigated in death receptor-mediated hepatocyte apoptosis.…”

Opioid receptor blockade reduces Fas-induced hepatitis in mice

“…13,19,20 However, the demonstration that glucocorticoids as well as ␤-adrenergic agonists protected hepatocytes from apoptosis [21][22][23][24] was incompatible with an indirect mechanism involving neutralization by naltrexone of central opioid receptors. This presumption was attested by investigating the potency of naloxone methiodide, an antagonist of all the three opioid receptor subclasses unable to cross the blood-brain barrier, to protect mice against Fas-induced hepatitis.…”

Glucocorticoid signalling affects pancreatic development through both direct and indirect effects

“…However, glucocorticoid levels have already been reported to play a role in the apoptotic process of various endodermally derived cell types. More precisely, it has been shown that dexamethasone treatment leads to an increased production of the anti-apoptotic factors Bcl2, BclxL or cFLIP (an inhibitor of caspase-8) in human and rat hepatocytes [35], or c-IAP2 (cellular inhibitor of apoptosis 2) in human lung epithelial cells [36]. Moreover, Wistar Bonn/Kobori rats, which spontaneously develop chronic pancreatitis, have a decreased pancreatic weight, low endogenous corticosterone levels and increased acinar cell apoptosis [37].…”