Glucocorticoid-mediated attenuation of the hsp70 response in trout hepatocytes involves the proteasome

The physiological implication of elevated cortisol levels on cellular heat-shock protein 70 (hsp70) response was examined using primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Trout hepatocytes treated with cortisol, the predominant glucocorticoid in teleosts, responded to the heat shock (+15 degrees C for 1 h) with a significant drop in hsp70 accumulation over a 24-h recovery period. [(35)S]methionine incorporation and pulse-chase studies confirmed that this cortisol impact was due to decreased hsp70 synthesis and not enhanced protein breakdown. Cortisol also significantly decreased glucocorticoid receptor (GR) expression in trout hepatocytes. This receptor downregulation was inhibited by the proteasomal inhibitors, lactacystin and MG-132, implying a role for the proteasome in GR downregulation by cortisol. Inhibiting the proteasome did not significantly modify heat-induced hsp70 accumulation in the absence of cortisol but significantly elevated hsp70 expression in the presence of cortisol in heat-shocked trout hepatocytes. Taken together, our results suggest proteasome-mediated GR degradation as a mechanism for the attenuation of hsp70 response by cortisol in heat-shocked hepatocytes.     

Modulation of Ah receptor and CYP1A1 expression by alpha-naphthoflavone in rainbow trout hepatocytes

The objective of this study was to evaluate whether alpha-naphthoflavone (ANF) modulates aryl hydrocarbon receptor (AhR) signaling in rainbow trout (Oncorhynchus mykiss). AhR and cytochrome P450 1A1 (CYP1A1) protein and mRNA content were used as indictors of AhR signaling. Primary culture of rainbow trout hepatocytes were exposed to different concentrations of ANF (10(-9)-10(-5) M), while beta-naphthoflavone (BNF 10(-10)-10(-6) M) and a combination of ANF and BNF were used to elucidate the impact of ANF on AhR signaling. ANF increased AhR and CYP1A1 protein expression in a concentration-related manner; the maximal induction was about 50% that of BNF. Despite the differences in protein content between ANF and BNF stimulation, the maximal AhR and CYP1A1 mRNA abundance seen with the high concentrations of ANF and BNF were similar. ANF significantly decreased ( approximately 50%) BNF-induced AhR protein expression (only at 10(-9) M), but not CYP1A1 protein and gene expression. In addition, ANF at a sub-maximal concentration (10(-7) M) did not affect BNF-induced AhR protein content, but increased the sensitivity of hepatocytes to BNF-mediated CYP1A1 protein expression. Taken together, the mode of action of ANF appears similar to BNF, including modulation of AhR expression and activation of AhR-mediated signaling in rainbow trout hepatocytes. Overall, ANF is not only a partial AhR agonist, but may also modify BNF-mediated AhR signaling in trout hepatocytes.     

Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21^Waf1/Cip1 mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21^Waf1/Cip1 induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.     

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin

The ubiquitin–proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.     

2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes

We report here a novel observation that 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) induced predominantly cytochrome P4501A1 (CYP1A1) in rat hepatocytes and predominantly CYP1A2 in human hepatocytes. As part of our research program to evaluate species-differences in response to CYP inducers, we studied the effects of TCDD on CYP1A activity, protein, and gene expression in primary cultures of rat and human hepatocytes. TCDD was found to induce CYP1A activity, measured as ethoxyresorufin-O-deethylase (EROD) activity, in both rat and human hepatocytes. TCDD induction of EROD activity in human hepatocytes (2-5 fold of concurrent solvent control), was significantly lower than that found in rat hepatocytes ( 20-fold of concurrent solvent control). Two structural analogs of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF), were also evaluated. As observed for TCDD, human hepatocytes consistently showed a lower response than rat hepatocytes. As most TCDD-related effects are believed to be mediated via binding of the TCDD-Ah receptor (AhR) complex to DNA, nuclear AhR levels were measured in rat and human hepatocytes after TCDD treatment. We found that the nuclear AhR levels in TCDD-treated rat hepatocytes were approximately 4 times higher than found in TCDD-treated human hepatocytes. However, the estimated binding affinity of [3H]TCDD to nuclear AhR from rat hepatocytes was similar. The species difference in response to TCDD was further evaluated by analysis of CYP1A1 and CYP1A2 mRNA levels using Northern analysis, and P4501A1 and 1A2 protein levels using Western immunoblotting. Results showed that, at both gene expression and protein levels, TCDD induced predominantly CYP1A1 in rat hepatocytes and CYP1A2 in human hepatocytes.     

The Ubiquitin–Proteasome System and Cellular Proliferation and Regulation in Osteoblastic Cells

The 26S proteasome is the macromolecular assembly that mediates ATP- and ubiquitin-dependent extralysosomal intracellular protein degradation in eukaryotes. However, its contribution to the regulation of osteoblast proliferation and hormonal regulation remains poorly defined. Treating osteoblasts with MG-132 or lactacystin (membrane-permeable proteasome inhibitors) attenuates proliferation. Three proteasome activities (peptidylglutamyl–peptide bond hydrolase-, chymotrypsin-, and trypsin-like) were detected in osteoblasts. Catabolic doses of PTH stim-ulated these activities, and cotreatment with PTH and MG-132 blocked stimulation. The proteasome α- and β-subunits, polyubiquitins, and large ubiquitin–protein conjugates were detected by Western blotting. A 90-min treatment with 10 nM PTH had no effect on the amount of proteasome α or β subunit protein, but increased the relative amount of large ubiquitin-protein conjugates by 200%. MG-132 inhibited deubiquitination of large ubiquitin–protein conjugates. The protein kinase A inhibitor SQ22536 blocked much of the PTH-induced stimulation of MCP activities, while dibutyryl cAMP stimulated it, suggesting that protein kinase A-dependent phosphorylation is important in PTH stimulation of proteasome activities. In conclusion, the ubiquitin–proteasome system is essential for osteoblast proliferation under control and PTH-treated conditions. PTH mediates its metabolic effects on the osteoblast, in part, by enhancing ubiquitinylation of protein substrates and stimulating three major proteasome activities by a cAMP-dependent mechanism.     

Characterization of the AhR-hsp90-XAP2 core complex and the role of the immunophilin-related protein XAP2 in AhR stabilization.

The unliganded aryl hydrocarbon receptor (AhR) exists in the cytoplasm in a tetrameric 9S core complex, consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2), an immunophilin-related protein sharing homologous regions with FKBP12 and FKBP52. Interactions between the recently identified XAP2 subunit and other members of the unliganded AhR complex and its precise role in the AhR signal transduction pathway are presently unknown. Mapping studies indicate that XAP2 requires the PAS, hsp90, and ligand binding domain(s) of the AhR for binding, and that both proteins directly interact in the absence of hsp90. XAP2 is also able to interact with hsp90 complexes in the absence of the AhR, and C-terminal sequences of XAP2 are required for this interaction. XAP2 binds to the C-terminal end of hsp90, which contains a tetratricopeptide repeat domain acceptor site, whereas the AhR binds to a domain in the middle of hsp90. XAP2 was not found to be associated with the AhR-Arnt heterocomplex either in vitro or in nuclear extracts isolated from Hepa 1 cells treated with TCDD. Transient expression of XAP2 in COS-1 cells resulted in enhanced cytosolic AhR levels, suggesting a role for XAP2 in regulating the rate of AhR turnover.     

Regulation of the C-X-C chemokine,mob-1, gene expression in primary rat hepatocytes

The chemokine, mob-1, is involved in inflammatory and immune responses and may be an important mediator of the inflammatory response in the liver. Here, we investigated the upstream signal pathways that could be involved in the regulation of mob-1 expression. We have found that in primary rat hepatocytes the isolation and subsequent culture of these cells induced mob-1 expression. A similar induction of mob-1 mRNA was observed when the hepatocytes were stimulated with interferon-gamma (IFN-gamma). When hepatocytes were stimulated with IFN-gamma or cytokine mixture (IFN-gamma, interleukin-1beta and tumour necrosis factor-alpha), c-Jun N-terminal kinase (JNK), p38 and extracellular-regulated kinase (ERK) were phosphorylated, suggesting an involvement of the mitogen-activated protein kinases (MAPK) in the induction of mob-1 expression. The p38 kinase inhibitor, SB 203580, and the NF-kappaB inhibitor, MG-132, inhibited the induction of mob-1 mRNA and the effects were not additive. These results demonstrate that in primary rat hepatocytes the transient induction of mob-1 expression was regulated by p38 kinase and NF-kappaB through a common regulatory pathway.     

Binding of aryl hydrocarbon receptor (AhR) to AhR-interacting protein. The role of hsp90

The aryl hydrocarbon receptor (AhR) has been shown to interact with an immunophilin-like molecule known as AhR-interacting protein (AIP) and to enhance AhR function. We show here that AIP associates with AhR homologues from mouse and fish, which can bind ligands such as dioxin, but nonligand binding homologues from Caenorhabditis elegans or Drosophila do not bind to AIP. However, a minimal ligand-binding domain of the AhR is incapable of binding AIP. The binding of AIP to AhR in reticulocyte lysate shows several of the characteristics of an hsp90-dependent process, including sensitivity to geldanamycin and temperature and a requirement for ATP or nonhydrolyzable analogues. Purified AIP binds to the C terminus of hsp90, and mutation of a conserved basic residue in the tetratricopeptide repeats of AIP (K266A, analogous to K97A in protein phosphatase 5) abolishes binding to hsp90. Mutation of K266A in AIP reduces binding to AhR by 75-80%; the geldanamycin sensitivity of this complex shows that AhR stabilizes the AIP-hsp90-AhR complex. The alpha-helical C terminus of AIP, which is outside the tetratricopeptide repeat domain, is absolutely required for binding to AhR as shown by deletions of the C-terminal 5 amino acids or alanine-scanning mutagenesis, but it is not required for binding of AIP to hsp90. The data support a model where 1) AIP binds to both hsp90 and AhR; 2) hsp90 is required for AhR-AIP binding; and 3) the binding of AhR to AIP stabilizes the AIP-hsp90-AhR complex.     

Osmotic regulation of MG-132-induced MAP-kinase phosphatase MKP-1 expression in H4IIE rat hepatoma cells.

BACKGROUND/AIMS: Proteasome inhibitors such as MG-132 are considered as potential therapeutical tools in different clinical settings. The dual specificity MAP-kinase phosphatase MKP-1 plays a role in balancing signals mediating cell death or survival. Here the effect of cell hydration on MG-132-induced MKP-1 expression was investigated in H4IIE rat hepatoma cells. RESULTS: Hyperosmolarity (405mosmol/l) increased MKP-1 expression by MG-132, which was accompanied by an induction of c-Fos, c-Jun, cJun Ser73 phosphorylation, and AP-1 DNA binding. MKP-1 induction by MG-132 plus hyperosmolarity was sensitive to inhibition of p38(MAPK) and c-Jun-N-terminal kinases (JNKs) but not extracellular signal-regulated kinases Erk-1/Erk-2, and was accompanied by a decline of MAP-kinase activities. Although hyperosmolarity increased overall protein ubiquitination in presence of MG-132, ubiquitination of MKP-1 was found under normo-, but not hyperosmotic conditions. Hyperosmolarity also enabled MG-132 to induce poly-ADP-ribose polymerase (PARP) cleavage which was sensitive to inhibition of p38(MAPK) and JNKs but not Erk-1/Erk-2. PARP cleavage and caspase-3 activation in H4IIE cells treated with hyperosmolarity plus MG-132 was further increased by vanadate, consistent with a contribution of MKP-1 to counterbalance proapoptotic MAP-kinase signals. CONCLUSION: The findings suggest that among other factors cell hydration critically determines the cellular response to proteasome inhibitors.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of ³⁵S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.