Matrix-dependent regulation of AKT in Hepsin-overexpressing PC3 prostate cancer cells.

The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox)-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser(473), which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases.

Age-related decrease in proteasome expression contributes to defective nuclear factor-kappaB activation during hepatic ischemia/reperfusion.

Hepatic ischemia/reperfusion (I/R) leads to liver injury and dysfunction through the initiation of a biphasic inflammatory response that is regulated by the transcription factor nuclear factor kappaB (NF-kappaB). We have previously shown that there is an age-dependent difference in the injury response to hepatic I/R in mice that correlates with divergent activation of NF-kappaB such that young mice have greater NF-kappaB activation, but less injury than old mice. In this study, we investigated the mechanism by which age alters the activation of NF-kappaB in the liver during I/R. Young (4-5 weeks) and old (12-14 months) mice underwent partial hepatic I/R. Livers were obtained for RNA microarray analysis and protein expression assays. Using microarray analysis, we identified age-dependent differences in the expression of genes related to protein ubiquitinylation and the proteasome. In old mice, genes that are involved in the ubiquitin-proteasome pathway were significantly down-regulated during I/R. Consistent with these findings, expression of a critical proteasome subunit, non-adenosine triphosphatase 4 (PSMD4), was reduced in old mice. Expression of the NF-kappaB inhibitory protein, IkappaB alpha, was increased in old mice and was greatly phosphorylated and ubiquitinylated. The data provide strong evidence that the age-related defect in hepatic NF-kappaB signaling during I/R is a result of decreased expression of PSMD4, a proteasome subunit responsible for recognition and recruitment of ubiquitinylated substrates to the proteasome. It appears that decreased PSMD4 expression prevents recruitment of phosphorylated and ubiquitinylated IkappaB alpha to the proteasome, resulting in a defect in NF-kappaB activation.

Peroxiredoxin-6 protects against mitochondrial dysfunction and liver injury during ischemia-reperfusion in mice.

Hepatic ischemia-reperfusion (I/R) injury is an important complication of liver surgery and transplantation. Mitochondrial function is central to this injury. To examine alterations in mitochondrial function during I/R, we assessed the mitochondrial proteome in C57Bl/6 mice. Proteomic analysis of liver mitochondria revealed 234 proteins with significantly altered expression after I/R. From these, 13 proteins with the greatest expression differences were identified. One of these proteins, peroxiredoxin-6 (Prdx6), has never before been described in mitochondria. In hepatocytes from sham-operated mice, Prdx6 expression was found exclusively in the cytoplasm. After ischemia or I/R, Prdx6 expression disappeared from the cytoplasm and appeared in the mitochondria, suggesting mitochondrial trafficking. To explore the functional role of Prdx6 in hepatic I/R injury, wild-type and Prdx6-knockout mice were subjected to I/R injury. Prdx6-knockout mice had significantly more hepatocellular injury compared with wild-type mice. Interestingly, the increased injury in Prdx6-knockout mice occurred despite reduced inflammation and was associated with increased mitochondrial generation of H(2)O(2) and dysfunction. The mitochondrial dysfunction appeared to be related to complex I of the electron transport chain. These data suggest that hepatocyte Prdx6 traffics to the mitochondria during I/R to limit mitochondrial dysfunction as a protective mechanism against hepatocellular injury.

Hepatocyte signaling through CXC chemokine receptor-2 is detrimental to liver recovery after ischemia/reperfusion in mice.

UNLABELLED: CXC chemokines and their receptor, CXC chemokine receptor-2 (CXCR2), are important components of the hepatic inflammatory response to ischemia/reperfusion (I/R). However, direct effects of CXC chemokines on hepatocytes during this response have not been studied. Wild-type and CXCR2(-/-) mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 96 hours of reperfusion. CXCR2(-/-) mice had significantly less liver injury at all reperfusion times compared with wild-type mice. Early neutrophil recruitment (12 hours) was diminished in CXCR2(-/-) mice, but within 24 hours it was the same as that of wild-type mice. Hepatocyte proliferation and regeneration was accelerated in CXCR2(-/-) mice compared with wild-type mice. These effects were associated with increased activation of nuclear factor kappaB and signal transducers and activators of transcription-3, despite there being no difference in the expression of proliferative factors such as tumor necrosis factor alpha, interleukin-6, and hepatocyte growth factor. To establish whether the accelerated proliferation and regeneration observed in CXCR2(-/-) mice was due to effects on hepatocytes rather than just a generalized decrease in acute inflammatory injury, mice were treated with the CXCR2 antagonist, SB225002, after neutrophil recruitment and injury were maximal (24 hours after reperfusion). SB225002 treatment increased hepatocyte proliferation and regeneration in a manner identical to that observed in CXCR2(-/-) mice. Treatment of primary wild-type hepatocytes with macrophage inflammatory protein-2 revealed that low concentrations protected against cell death, whereas high concentrations induced cell death. These effects were absent in hepatocytes from CXCR2(-/-) mice. CONCLUSION: Our data suggest that hepatocyte CXCR2 regulates proliferation and regeneration after I/R injury and reveal important differences in the role of this receptor in liver regeneration and repair induced under different conditions that may be related to ligand concentration.

Activation of hepatocytes by extracellular heat shock protein 72.

Heat shock protein (HSP) 72 is released by cells during stress and injury. HSP-72 also stimulates the release of cytokines in macrophages by binding to Toll-like receptors (TLR) 2 and 4. Circulating levels of HSP-72 increase during hepatic ischemia-reperfusion injury. The role of extracellular HSP-72 (eHSP-72) in the injury response to ischemia-reperfusion is unknown. Therefore, the objective of the present study was to determine whether eHSP-72 has any direct effects on hepatocytes. Primary mouse hepatocytes were treated with purified human recombinant HSP-72. Conditioned media were evaluated by ELISA for the cytokines, TNF-alpha, IL-6, and macrophage inflammatory protein 2 (MIP-2). Stimulation of hepatocytes with eHSP-72 did not induce production of TNFalpha or IL-6 but resulted in dose-dependent increases in MIP-2 production. To evaluate the pathway responsible for this response, expression of TLR2 and TLR4 was confirmed on hepatocytes by immunohistochemistry. Hepatocyte production of MIP-2 was significantly decreased in hepatocytes obtained from TLR2 or TLR4 knockout mice. MIP-2 production was found to be partially dependent on NF-kappaB because inhibition of NF-kappaB with Bay 11-7085 significantly decreased eHSP-72-induced MIP-2 production. Inhibitors of p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase had no effect on production of MIP-2 induced by eHSP-72. The data suggest that eHSP-72 binds to TLR2 and TLR4 on hepatocytes and signals through NF-kappaB to increase MIP-2 production. The fact that eHSP-72 did not increase TNF-alpha or IL-6 production may be indicative of a highly regulated signaling pathway downstream from TLR.

Activation of peroxisome proliferator-activated receptor-gamma during hepatic ischemia is age-dependent.

Hepatic ischemia/reperfusion injury is a complication of liver surgery, transplantation, and shock and is known to be age-dependent. Our laboratory has recently shown that peroxisome proliferator-activated receptor-gamma (PPARgamma) is down-regulated during hepatic ischemia and that this exacerbates injury. Here we examined whether activation of PPARgamma during ischemia was age-dependent. Male mice of different ages (young: 4-5 weeks; adult: 10-12 weeks; old: 10-12 months) were subjected to up to 90 min of hepatic ischemia. PPARgamma activation occurred throughout ischemia in young mice, whereas activation in adult and old mice was lost after 30 min. No significant differences were noted in PPARgamma ligand expression among the age groups. However, in young mice we observed a predominance of PPARgamma1 in the nucleus, whereas in old mice this isoform remained largely in the cytoplasm. Finally, the degree of PPARgamma activation was associated with autophagy in the liver, a mechanism of self-preservation. PPARgamma activation is prolonged in young mice as compared to older mice. This appears to be mediated by a selective retention of PPARgamma1 in the nucleus and is associated with increased autophagy. The data suggest that PPARgamma activation is an important component of the age-dependent response to hepatic ischemia/reperfusion injury.

Peroxisome proliferator-activated receptor-gamma protects against hepatic ischemia/reperfusion injury in mice.

UNLABELLED: The function of peroxisome proliferator-activated receptor-gamma (PPARgamma) in hepatic inflammation and injury is unclear. In this study, we sought to determine the role of PPARgamma in hepatic ischemia/reperfusion injury in mice. Male mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 8 hours of reperfusion. PPARgamma was found to be constitutively activated in hepatocytes but not in nonparenchymal cells. Upon induction of ischemia, hepatic PPARgamma activation rapidly decreased and remained suppressed throughout the 8-hour reperfusion period. This reduced activation was not a result of decreased protein availability as hepatic nuclear PPARgamma, retinoid X receptor-alpha (RXRalpha), and PPARgamma/RXRalpha heterodimer expression was maintained. Accompanying the decrease in PPARgamma activation was a decrease in the expression of the natural ligand 15-deoxy-Delta(12,14)-prostaglandin J(2). This was associated with reduced interaction of PPARgamma and the coactivator, p300. To determine whether PPARgamma activation is hepatoprotective during hepatic ischemia/reperfusion injury, mice were treated with the PPARgamma agonists, rosiglitazone and connecting peptide. These treatments increased PPARgamma activation and reduced liver injury compared to untreated mice. Furthermore, PPARgamma-deficient mice had more liver injury after ischemia/reperfusion than their wild-type counterparts. CONCLUSION: These data suggest that PPARgamma is an important endogenous regulator of, and potential therapeutic target for, ischemic liver injury.

The role of the casein kinase 1 (CK1) family in different signaling pathways linked to cancer development.

The members of the casein kinase 1 (CK1) family are highly conserved and are expressed in many eukaryotes ranging from yeast to humans. Mammalian CK1 isoforms (alpha, beta, gamma, delta, epsilon) and their splice variants are involved in diverse cellular processes including membrane trafficking, circadian rhythm, cell cycle progression, chromosome segregation, apoptosis and cellular differentiation. Mutations and deregulation of CK1 expression and activity has been linked to various diseases including neurodegenerative disorders such as Alzheimer's and Parkinson's disease, sleeping disorders and proliferative diseases such as cancer. In this review, we summarize the functions of CK1 and outline the participation of CK1 in signal transduction pathways linked to cancer development.