Structural and functional changes in the alpha A-crystallin R116C mutant in hereditary cataracts

alpha-Crystallin, the major protein component of vertebrate lenses, forms a large complex comprised of two homologous subunits, alphaA- and alphaB-crystallin. It has the ability to suppress stress-induced protein aggregation in vitro, bind saturably to lens plasma membranes, and aid in light refraction through short-range ordering. Recently, a missense mutation in alphaA-crystallin that changes arginine 116 to a cysteine residue (R116C) was genetically linked to one form of autosomal dominant congenital cataracts. This point mutation is reported to cause structural alterations at many levels as well as a 4-fold reduction in chaperone-like activity. To extend these findings, we examined the quaternary stability of the alphaA R116C mutant protein and its effect on chaperone-like activity, subunit exchange, and membrane association. Homocomplexes of mutant subunits become highly polydisperse following incubation at 37 degrees C, reflecting the likely in vivo distribution of the complexes. Chaperone-like activity of the alphaA R116C mutant is approximately 4-fold lower than wild type, whether measured before or after conversion to a polydisperse population with incubation. alphaA R116C complexes also have a 4-fold reduced ability to exchange subunits with wild-type complexes. Finally, membrane binding capacity measurements of mutant subunits showed a 10-fold increase over wild type. Our results, in conjunction with previous reports, suggest that the changes in complex polydispersity, the reduction of subunit exchange, and increased membrane binding capacity are all potential factors in the pathogenesis of alphaA R116C associated congenital cataracts.     

Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function

An autosomal dominant congenital cataract in human is associated with mutation of Arg-116 to Cys (R116C) in alpha A-crystallin. To investigate the molecular basis of cataract formation, rat alpha A-crystallin cDNA was cloned into pET-23d(+), and the site-directed mutants S142C (similar to wild-type human alpha A) and R116C/S142C or R116C (similar to human R116C variant) were generated. These were expressed in E. coli and the recombinant alpha A-crystallins purified by Sephacryl size-exclusion chromatography. The chaperone-like function of mutant R116C determined at 37 degrees C with insulin and alcohol dehydrogenase as target proteins was about 40% lower than those of wild-type and mutant S142C. Based on size-exclusion chromatography data, the oligomeric size of the R116C mutant was about 2000 kDa at 25 degrees C, 1400 kDa at 37 degrees C, and 900 kDa at 45 degrees C. In comparison, alpha A-wild-type and alpha A-S142C ranged from 477 to 581 kDa. Heat stability studies corroborated the effect of temperature on the dynamic quaternary structure of the R116C mutant. Circular dichroism spectra showed secondary and tertiary structural changes, and ANS fluorescence spectra showed loss of surface hydrophobicity in the R116C mutant. These findings suggest that the molecular basis for the congenital cataract with the alpha A-R116C mutation is due to the generation of a highly oligomerized alpha A-crystallin having a modified structure and decreased chaperone-like function.     

Folacin C60 derivative exerts a protective activity against oxidative stress-induced apoptosis in rat pheochromocytoma cells

In the present study, we describe the synthesis and characterization of a novel folacin C₆₀ derivative. The compound was analyzed by FT-IR, ¹H NMR, ¹³C NMR, LC–MS and elemental analysis. This water soluble fullerene derivative was able to scavenge both superoxide and hydroxyl radical with biocompatibility. Rat pheochromocytoma (PC12) cells treated with hydrogen peroxide underwent cytotoxicity and apoptotic death determined by MTT assay and flow cytometry analysis. As a novel derivative of C₆₀, the folacin C₆₀ derivative self-assembled to form spherical aggregates in H₂O. Because the compound was amphiphilic, it could penetrate the cell membrane and play its distinguished role in protecting PC12 cells against hydrogen peroxide-induced cytotoxicity. The results suggest that folacin C₆₀ derivative has the potential to prevent oxidative stress-induced cell death without evident toxicity.     

C60 carboxyfullerene exerts a protective activity against oxidative stress-induced apoptosis in human peripheral blood mononuclear cells

C60 carboxyfullerene is a novel buckminsterfullerene-derived compound that behaves as a free-radical scavenger. In the present report, we investigated whether this drug exerts a protective activity against oxidative stress-induced apoptosis. Human peripheral blood mononuclear cells (PBMCs) were challenged by 2-deoxy-d-ribose (dRib) or TNF-alpha plus cycloheximide as agents that trigger apoptosis by interfering with the redox status of cell and mitochondrial membrane potential. We found that carboxyfullerene was able to protect quiescent PBMCs from apoptosis caused either by 2-deoxy-d-ribose or TNF-alpha plus cycloheximide by a mechanism partially involving the mitochondrial membrane potential integrity, known to be associated with early stages of apoptosis. These results represent the first indication for a target activity of buckminsterfullerenes on cells of the immune system and their mitochondria.     

Differential protective activity of alpha A- and alphaB-crystallin in lens epithelial cells

alphaA- and alphaB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of alphaA- and alphaB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of alphaA- and alphaB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of alphaA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of alphaB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor alpha. To further study the relative protective activities of alphaA- and alphaB-crystallins, we created a cell line derived from alphaA-/-alphaB-/- double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line alphaAalphaBKO1 derived from alphaA/alphaB double knockout cells was transfected with alphaA- or alphaB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either alphaA-crystallin or alphaB-crystallin were isolated. The ability of alphaA- or alphaB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of alphaA-crystallin was two to three-fold higher than that of alphaB-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of alphaA- and alphaB-crystallin. Since the expression of alphaA-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.     

bFGF suppresses serum-deprivation-induced apoptosis in a human lens epithelial cell line.

There is increasing evidence that basic fibroblast growth factor (bFGF) plays an important role in cell proliferation, differentiation, and survival in various systems. In the eye, although a truncated, dominant negative bFGF receptor in transgenic mice induced defective lens development and caused lens fiber cells to display characteristics of apoptosis, there is little direct evidence of the effect of bFGF on lens epithelial cell apoptosis. Our study examines the effects of bFGF on programmed cell death induced by serum deprivation using a human lens epithelial cell line. Cells supplemented with 20% fetal bovine serum were used as normal controls. Over a period of 7 days, the addition of 100 ng/ml bFGF effectively suppressed serum-deprived apoptosis. The expression of gamma-crystallin and major intrinsic protein, which are markers of lens cell differentiation, was not detected. Also there was no significant difference in cell proliferation between serum-deprived cells with or without bFGF. ICE (caspase-1) was expressed under both the conditions, but the level of expression between the two groups was not substantially different. bcl-2 and c-myc were upregulated only in bFGF-treated cells. Thus we speculate that the inhibitory effect of bFGF on apoptosis is through the upregulation of the inhibitor of apoptosis, instead of downregulation of the initiator. This effect appears to be independent of lens cell differentiation and proliferation.     

Mechanism of small heat shock protein function in vivo: a knock-in mouse model demonstrates that the R49C mutation in alpha A-crystallin enhances protein insolubility and cell death

alphaA-crystallin (Cryaa/HSPB4) is a small heat shock protein and molecular chaperone that prevents nonspecific aggregation of denaturing proteins. Several point mutations in the alphaA-crystallin gene cause congenital human cataracts by unknown mechanisms. We took a novel approach to investigate the molecular mechanism of cataract formation in vivo by creating gene knock-in mice expressing the arginine 49 to cysteine mutation (R49C) in alphaA-crystallin (alphaA-R49C). This mutation has been linked with autosomal dominant hereditary cataracts in a four-generation Caucasian family. Homologous recombination in embryonic stem cells was performed using a plasmid containing the C to T transition in exon 1 of the cryaa gene. alphaA-R49C heterozygosity led to early cataracts characterized by nuclear opacities. Unexpectedly, alphaA-R49C homozygosity led to small eye phenotype and severe cataracts at birth. Wild type littermates did not show these abnormalities. Lens fiber cells of alphaA-R49C homozygous mice displayed an increase in cell death by apoptosis mediated by a 5-fold decrease in phosphorylated Bad, an anti-apoptotic protein, but an increase in Bcl-2 expression. However, proliferation measured by in vivo bromodeoxyuridine labeling did not decline. The alphaA-R49C heterozygous and homozygous knock-in lenses demonstrated an increase in insoluble alphaA-crystallin and alphaB-crystallin and a surprising increase in expression of cytoplasmic gamma-crystallin, whereas no changes in beta-crystallin were observed. Co-immunoprecipitation analysis showed increased interaction between alphaA-crystallin and lens substrate proteins in the heterozygous knock-in lenses. To our knowledge this is the first knock-in mouse model for a crystallin mutation causing hereditary human cataract and establishes that alphaA-R49C promotes protein insolubility and cell death in vivo.     

Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-crystallin gene

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.     

Identification of a protective role for protein phosphatase 1cgamma1 against oxidative stress-induced vascular smooth muscle cell apoptosis

The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1cgamma1 (catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1cgamma1 activity and expression. Depletion of PP1cgamma1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1cgamma1 overexpression abrogated DMNQ-induced JNK1 activity, p53 Ser(15) phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1cgamma1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1cgamma1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis.     

Caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis

Phosphorylation and dephosphorylation are important cellular events regulating major metabolic activities such as signal transduction, gene expression, cell cycle progression, and apoptosis. It is well documented that okadaic acid, a potent inhibitor of protein phosphatase-1 (PP-1) and -2A (PP-2A), can induce apoptosis in a variety of cell lines. Our recent studies have revealed that in the immortal rabbit lens epithelial cell line, N/N1003A, inhibition of PP-1, but not PP-2A, leads to rapid apoptosis of the lens epithelial cells. This induction of cell death is associated with up-regulated expression of a set of genes, including the tumor-suppressor gene, p53, and the proapoptotic gene, bax. In the present study, we demonstrate that inhibition of PP-1 by okadaic acid in the primary cultures of rat lens epithelial cells also leads to apoptotic death. Moreover, we show that the cysteine protease, caspase-3, is important in the execution of okadaic acid-induced apoptosis. Treatment of the primary cultures of rat lens epithelial cells with 100 nM okadaic acid up-regulates expression of caspase-3 at the mRNA, protein, and enzyme activity levels. Inhibition of the caspase-3 activity with a chemically synthesized inhibitor prevents okadaic acid-induced apoptosis in rat lens epithelial cells. Similar results are also observed in the immortal cell line N/N1003A. Furthermore, stable expression of the mouse gene encoding lens alphaB crystallin inhibits okadaic acid-induced apoptosis, and this inhibition is associated with repression of the okadaic acid-induced up-regulation of caspase-3 activity. Taken together, these results demonstrate that caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis.     

Developmental analysis of ocular morphogenesis in alpha A-crystallin/diphtheria toxin transgenic mice undergoing ablation of the lens.

The role of the lens in early eye development was examined in transgenic mice carrying the cytotoxic diphtheria toxin A gene driven by hamster alpha A-crystallin promoter sequences. Mice hemizygous for this construct are microphthalmic and contain a vacuolated and highly disorganized lens, whereas adult homozygous mice are completely ablated of the lens and lack a pupil, aqueous and posterior chamber, vitreous humor, iris, and ciliary body and show extensive convolution of the sensory retina. Developmental analysis of animals homozygous for the transgene revealed that the optic cup and lens vesicle form normally and that ablation of the lens occurs as a gradual degenerative process beginning between Days 12 and 13 of gestation. Degeneration of the lens vesicle coincides with retarded growth and development of the neuroretina, sclera, and cornea. The anterior lip of the optic cup fails to differentiate into the normal epithelium of the iris and ciliary body and the vitreous body does not develop. Although the retinal layers apparently form normally, retinal folding becomes prominent following lens degeneration. These results suggest that development of a functional lens from Embryonic Day 12.5 onward is critical for formation of the ciliary epithelium, iris, and vitreous body, as well as for appropriate growth, development, and maintenance of morphology of the retina, cornea, sclera, and optic nerve. Our results also provide information on the time course of DT-A-mediated cell destruction in vivo and are discussed in context with previous lens ablation studies and the importance of developmental analysis for interpretation of the extent to which morphogenetic aberrations are concurrent with or secondary to genetic ablation of the target tissue.     

MicroRNA-216b inhibits heat stress-induced cell apoptosis by targeting Fas in bovine mammary epithelial cells

Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.     

The roles of thioredoxin in protection against oxidative stress-induced apoptosis in SH-SY5Y cells.

Using models of serum deprivation and 1-methyl-4-phenylpyridinium (MPP(+)), we investigated the mechanism by which thioredoxin (Trx) exerts its antiapoptotic protection in human neuroblastoma cells (SH-SY5Y) and preconditioning-induced neuroprotection. We showed that SH-SY5Y cells are highly sensitive to oxidative stress and responsive to both extracellularly administered and preconditioning-induced Trx. Serum deprivation and MPP(+) produced an elevation in the hydroxyl radicals, malondialdehyde and 4-hydroxy-2,3-nonenal (HNE), causing the cells to undergo mitochondria-mediated apoptosis. Trx in the submicromolar range blocked the observed apoptosis via a multiphasic protection mechanism that includes the suppression of cytochrome c release (most likely via the induction of Bcl-2), the inhibition of procaspase-9 and procaspase-3 activation, and the elevated level of Mn-SOD. The reduced form of Trx suppresses the serum-free-induced hydroxyl radicals, lipid peroxidation, and apoptosis, indicating that H(2)O(2) is removed by Trx peroxidase. The participation of Trx in preconditioning-induced neuroprotection is supported by the observation that inhibition of Trx synthesis with antisense oligonucleotides or of Trx reductase drastically reduced the hormesis effect. This effect of Trx-mediated hormesis against oxidative stress-induced apoptosis is striking. It induced a 30-fold shift in LD(50) in the MPP(+)-induced neurotoxicity.     

The alpha 6 beta 4 integrin and epithelial cell migration.

Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.     

Role of hsp105 in protection against stress-induced apoptosis in neuronal PC12 cells.

hsp105alpha is a stress protein characteristically highly expressed in the brain compared with other tissues in mammals. Here, to examine whether hsp105alpha plays a pivotal role in the nervous system, we tested the capability of hsp105alpha to protect against apoptosis in rat neuronal PC12 cells. Various stress treatments such as serum deprivation, heat shock, hydrogen peroxide, etoposide, and actinomycin D induced apoptosis in PC12 cells with characteristic shrinking of nuclei and chromatin. However, PC12 cells that constitutively overexpressed mouse hsp105alpha exhibited a strong protective effect against apoptosis induced by these stress treatments. Cleavage of poly(ADP-ribose) polymerase induced in PC12 cells by these treatments was inhibited in the constitutively overexpressed hsp105alpha cells. Furthermore, c-Jun N-terminal kinase (JNK) was activated in the cells treated with heat shock but not other treatments, and the heat-induced JNK activation was inhibited by the constitutive expression of hsp105alpha.Thus, hsp105alpha prevents not only heat-induced apoptosis by inhibiting JNK activation, but also prevents the apoptosis induced by other stressors through different pathways, and may play important roles in neuronal protection.     

The alphaA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with alphaB-crystallin.

An autosomal dominant congenital cataract in humans is associated with mutation of Arg-116 to Cys in alphaA-crystallin (alphaA-R116C). The chaperone activity and biophysical properties of reconstituted alpha-crystallin from different proportions of wild-type alphaB-crystallin (alphaB-wt) and alphaA-R116C-crystallin were studied by gel permeation chromatography, SDS-polyacrylamide gel electrophoresis, and fluorescence and circular dichroism spectroscopy and compared with those of reconstituted alpha-crystallin from alphaB-wt and wild-type alphaA-crystallin (alphaA-wt). The reconstituted alpha-crystallin containing alphaA-R116C and alphaB-wt had a higher molecular mass, a higher thermal sensitivity to exposition of Trp side chains, fewer available hydrophobic surfaces, and lower chaperone activity than the alpha-crystallin containing alphaA-wt and alphaB-wt. The secondary structure exhibited very small changes, whereas the tertiary structure was distinctly different for alpha-crystallin formed from alphaA-R116C and alphaB-wt. Most importantly, subunit exchange studies by fluorescence resonance energy transfer showed that alphaA-R116C forms heteroaggregates faster than alphaA-wt with alphaB-wt, and the reconstituted alpha-crystallins were true heteroaggregates of two interacting subunits. These findings suggest that the molecular basis for the congenital cataract with the alphaA-R116C mutation is the formation of highly oligomerized heteroaggregates of alpha-crystallin with modified structure. However, contrary to the earlier conclusions based on the studies of homoaggregates, the loss in chaperone activity of the heteroaggregates having alphaA-R116C does not appear to be large enough to become the main factor in initiating cataract development in the affected individuals.     

Enhancement of oxidative stress-induced apoptosis by Hsp105alpha in mouse embryonal F9 cells.

Hsp105alpha is one of the major mammalian heat shock proteins that belongs to the HSP105/110 family, and is expressed at especially high levels in the brain as compared with other tissues in mammals. Previously, we showed that Hsp105alpha prevents stress-induced apoptosis in neuronal PC12 cells, and is a novel anti-apoptotic neuroprotective factor in the mammalian brain. On the other hand, we have also demonstrated that Hsp105alpha is expressed transiently at high levels during mouse embryogenesis and is found not only in various tissues but also in apoptotic cells. In the present study, to elucidate the role of Hsp105alpha during mouse embryogenesis, we established mouse embryonal F9 cell lines that constitutively over-express Hsp105alpha. Over-expression of Hsp105alpha enhanced hydrogen peroxide-induced apoptosis by enhancing the activation of caspase-3, poly(ADP-ribose)polymerase cleavage, cytochrome c release and activation of p38 mitogen-activated protein kinase (p38). Furthermore, oxidative stress-induced apoptosis was suppressed by SB202190, a potent inhibitor of p38, in F9 cells. These findings indicated that the activation of p38 is an essential step for apoptosis in F9 cells and that Hsp105alpha enhances activation of p38, release of cytochrome c and caspase activation. Hsp105alpha may play important roles in organogenesis, during which marked apoptosis occurs, by enhancing apoptosis during mouse embryogenesis.     

The essential role of PKCalpha in the protective effect of heat-shock pretreatment on TNFalpha-induced apoptosis in hepatic epithelial cell line

During sepsis, hepatic apoptosis occurred, which is associated with inactivation of PKCalpha and elevation of tumor necrosis factor-alpha (TNFalpha), an apoptosis trigger. Heat shock, accompanied by the increase of heat-shock protein (Hsp72), has been shown to exhibit a protective role on cell survival. However, Hsp72 was unable to express during sepsis when the apoptosis was markedly increased. We hypothesized that hepatic apoptosis during sepsis may be due to the failure to induce expression of Hsp72, which is activated by PKC-phosphorylated HSF. This study was designed to examine the role of PKCalpha in Hsp72 expression and the anti-apoptotic effect of Hsp72 on hepatic epithelial cells by analyzing a TNFalpha-induced apoptosis system. The following results were observed: (1) Hsp72 was highly expressed at 8 h after heat-shock treatment in a clone 9 hepatic epithelial cell line; (2) the protein expression of PKCalpha in membrane-associated fraction was decreased by TNFalpha treatment; (3) the TNFalpha-induced cell death, especially apoptosis, was diminished by heat-shock pretreatment; (4) in the presence of PKCalpha antisense, which blocks the PKCalpha resynthesis, no protective effect of heat-shock pretreatment was observed, and the protein expression of Hsp72 was significantly suppressed. These results suggest that PKCalpha plays a critical role in the expression of Hsp72, which subsequently protects against TNFalpha-induced hepatic apoptosis.