Hsp70i Expression Research Articles

HSP70iQ435A-Encoding DNA Repigments Vitiligo Lesions in Sinclair Swine

Human HSP70iQ435A carries a single amino-acid modification within the dendritic cell activating region and tolerizes dendritic cells invitro. The underlying DNA was used to prevent and treat disease in vitiligo mouse models through reduced dendritic cell activation and diminished skin T-cell infiltration, suggesting the same may be useful for patients. Physiologic differences between mouse and human skin then called for studies in large animals with human-like skin. We established the efficiency of DNA jet injection into swine skin before subcloning HSP70iQ435A into clinically suitable vector pUMVC3. Vitiligo lesions in Sinclair swine were treated with plasmid DNA to measure changes in depigmentation, T-cell infiltration, expression of HSP70i in skin, serum HSP70i, and anti-HSP70i serum titers. Remarkable repigmentation following HSP70iQ435A-encoding DNA treatment persisted throughout the 6-month follow-up period. Repigmentation was accompanied by an initial influx of T cells accompanied by increased CD4/CD8 ratios, waning by week 15. Melanocytes spanned the border of repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. Serum titer fluctuations were not treatment-associated. Importantly, treatment did not interfere with melanoma immunosurveillance. These data encourage clinical testing of HSP70iQ435A.

An inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating Hsp70 as a host antiviral target

An estimated three billion people are at risk of Dengue virus (DENV) infection worldwide and there are currently no approved therapeutic interventions for DENV infection. Due to the relatively small size of the DENV genome, DENV is reliant on host factors throughout the viral life cycle. The inducible form of Heat Shock Protein 70 (Hsp70i) has been implicated as a host factor in DENV pathogenesis, however the complete role remains to be elucidated. Here we further illustrate the importance of Hsp70i in dengue virus pathogenesis and describe the antiviral activity of the allosteric small molecule inhibitor that is selective for Hsp70i, called HS-72. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained. HS-72 works to reduce DENV infection by inhibiting the entry stage of the viral life cycle, through disrupting the association of Hsp70i with the DENV receptor complex. This work highlights Hsp70i as an antiviral target and HS-72 as a potential anti-DENV therapeutic agent.

Outcomes of lung cancer surgery after neo-adjuvant treatment: a National Surgical Quality Improvement Program analysis

METHODS: 26 dogs underwent bypass; temperature was raised to 42 C in 14 dogs (heat-shocked HS) or maintained at 37 C in 12 dogs (nonheat-shocked NHS). After 12 or 24 hours, heart rate (HR), left ventricular pressure (LVP), and peak +dP/dT were measured, as well as preload recruitable stroke work relationship (PRWA) throughout reperfusion. Myocardial expression of hsp70i was quantified by Western blots.

SIRT1 overexpression ameliorates a mouse model of SOD1-linked amyotrophic lateral sclerosis via HSF1/HSP70i chaperone system.

BackgroundDominant mutations in superoxide dismutase 1 (SOD1) cause degeneration of motor neurons in a subset of inherited amyotrophic lateral sclerosis (ALS). The pathogenetic process mediated by misfolded and/or aggregated mutant SOD1 polypeptides is hypothesized to be suppressed by protein refolding. This genetic study is aimed to test whether mutant SOD1-mediated ALS pathology recapitulated in mice could be alleviated by overexpressing a longevity-related deacetylase SIRT1 whose substrates include a transcription factor heat shock factor 1 (HSF1), the master regulator of the chaperone system.ResultsWe established a line of transgenic mice that chronically overexpress SIRT1 in the brain and spinal cord. While inducible HSP70 (HSP70i) was upregulated in the spinal cord of SIRT1 transgenic mice (PrP-Sirt1), no neurological and behavioral alterations were detected. To test hypothetical benefits of SIRT1 overexpression, we crossbred PrP-Sirt1 mice with two lines of ALS model mice: A high expression line that exhibits a severe phenotype (SOD1G93A-H) or a low expression line with a milder phenotype (SOD1G93A-L). The Sirt1 transgene conferred longer lifespan without altering the time of symptomatic onset in SOD1G93A-L. Biochemical analysis of the spinal cord revealed that SIRT1 induced HSP70i expression through deacetylation of HSF1 and that SOD1G93A-L/PrP-Sirt1 double transgenic mice contained less insoluble SOD1 than SOD1G93A-L mice. Parallel experiments showed that Sirt1 transgene could not rescue a more severe phenotype of SOD1G93A-H transgenic mice partly because their HSP70i level had peaked out.ConclusionsThe genetic supplementation of SIRT1 can ameliorate a mutant SOD1-linked ALS mouse model partly through the activation of the HSF1/HSP70i chaperone system. Future studies shall include testing potential benefits of pharmacological enhancement of the deacetylation activity of SIRT1 after the onset of the symptom.

COX-2 inhibition improves retinal function in rats' ischemic eyes.

Retinal ischemia is a relatively simple model for studies in pharmacological neuroprotective intervention. The role of cyclooxygenase (COX) enzymes in ischemic insult has been variously shown to either increase or decrease ischemic damage. The purpose of this study was to assess the role of COX-1 and COX-2 in rat retinal ischemic functional damage. Ischemia was achieved by elevating intraocular pressure for 60 min. White flash electroretinogram (ERG) was recorded by contact lens electrodes containing an integral light emitting diode source. ERG was recorded on post-ischemia (PI) days-1 (baseline), 1, 3, and 7. The b-wave amplitude, b-wave implicit time, and oscillatory potentials (OPs) were analyzed. The expression of COX-2 and HSP70i was assessed by Western analysis on day 1 PI. Ischemia caused attenuation of OPs, a decrease in the b-wave amplitude, and an increase in b-wave implicit time, accompanied by the increased expression of COX-2 and HSP70i proteins. Selective COX-2 inhibition markedly increased b-wave amplitude and enhanced retinal HSP70i induction, whereas COX-1 or nonselective and irreversible inhibition of both COX isoenzymes did not affect the retinal function or the expression of these proteins. High-dose aspirin prevented partial recovery from ischemic damage. Administration of a synthetic PGF2α analog, or a lipoxygenase inhibitor, had little effect on ischemic damage, but affected nonischemic ERG. COX-2 appears to mediate some of the ischemic retinal functional damage, possibly by inhibiting the induction of HSP-70i. We propose that selective COX-2 inhibitors may be useful in pathological conditions involving ischemic retinal insult.

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress

Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy, and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus, whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes.

The differential effects of acute vs. chronic stress and their combination on hippocampal parvalbumin and inducible heat shock protein 70 expression

The hippocampus plays a central role in stress-related mood disorders. The effects of acute vs. chronic stress on the integrity of hippocampal circuitry in influencing the vulnerability to, or resiliency against, neuronal injury are poorly understood. Here we investigated whether acute vs. chronic psychosocial isolation stress or a combination of the two (chronic stress followed by acute stress) influences the expression of the interneuronal marker parvalbumin (PV) and the chaperone-inducible heat shock protein 70 (Hsp70i) in different subregions of the hippocampus. Low levels of the Ca2+-binding protein (PV) may increase the vulnerability to neuronal injury, and Hsp70i represents an indicator of intense excitation-induced neuronal stress. Adult male Wistar rats were exposed to 2h of immobilization (IM) or cold (4°C) (acute stressors), 21d of social isolation (chronic stress), or a combination of both acute and chronic stress. Both chronic isolation and the combined stressors strongly decreased the PV-immunoreactive cells in the CA1, CA3 and dentate gyrus (DG) region of the hippocampus, while acute stress did not affect PV expression. The combination of acute and chronic stress induced a dramatic increase in Hsp70i expression in the DG, but Hsp70i expression was unaffected in acute and chronic stress alone. We also monitored serum corticosterone (CORT) levels as a neuroendocrine marker of the stress response. Acute stress increased CORT levels, while chronic isolation stress compromised hypothalamic–pituitary–adrenocortical (HPA) axis activity such that the normal stress response was impaired following subsequent acute stress. These results indicate that in contrast to acute stress, chronic isolation compromises the HPA axis and generates a considerable reduction in PV expression, representing a decrease in the calcium-buffering capacity and a putatively higher vulnerability of specific hippocampal interneurons to excitotoxic injury. The induction of Hsp70i expression in response to acute and chronic isolation reveals that neurons in the DG are particularly vulnerable to an acute stressor following a chronic perturbation of HPA activity.

PKC‐alpha regulates expression of the hsp70 promoter via its two proximal AP‐1 binding elements

Research has shown that PKC isozymes and heat shock proteins contribute to cardioprotection. Recent investigations into the interaction between these protein families revealed that PKC-alpha can confer protection against simulated ischemia by mediating the induction of Hsp70i. Interestingly, Hsp70i expression was found to be independent of the conventional heat shock transcription factor HSF-1. In an effort to elucidate a potentially novel mechanism, luciferase reporter assays were performed on chimeric rat Hsp70i promoter constructs transfected into myogenic H9c2 cells. Adenoviral overexpression of wild-type PKC-alpha demonstrates that inducibility of the rat Hsp70 promoter is preserved within 175 base pairs from the start of transcription. This segment contains two canonic AP-1 binding elements. Although both AP-1 sites retain modest induction in isolation, a more robust response occurs when present in tandem. Treatment with inhibitors targeting RhoA (C3 exoenzyme), ROCK (Y27693) and MEK (PD98059) suggest PKC-alpha transduces its signal through the raf-1/MEK/ERK pathway to upregulate c-Fos expression. These observations are supported by electrophoretic mobility shift assays confirming that c-Fos and c-Jun components bind to both the distal and proximal AP-1 elements. Potentially, these investigations may lead to new therapeutic approaches to harness the cardioprotective effects of hsp70.

Myocardial expression of heat shock protein 70i protects early postoperative right ventricular function in cyanotic tetralogy of Fallot

Right ventricular dysfunction occurs after tetralogy of Fallot repair and may relate to greater myocardial vulnerability to ischemia-reperfusion injury in cyanotic patients. The inducible form of heat shock protein 70 (HSP-70i), a molecular chaperone, is upregulated in response to cellular stress and limits myocardial injury against ischemia-reperfusion. We evaluated the myocardial expression of HSP-70i and its relation to right ventricular function and clinical outcome in patients with tetralogy of Fallot undergoing corrective surgery. Twenty patients with tetralogy of Fallot were studied: 10 cyanotic (group Cy) and 10 noncyanotic (group noCy). Western blot was used to quantify HSP-70i from resected right ventricular outflow tract myocardium at baseline and subsequent ischemic time. Biventricular function was quantified by tissue Doppler echocardiography and compared with that of 15 age-matched healthy children. Postoperative systemic perfusion was assessed by mixed venous oxygen saturation, oxygen extraction ratio, and lactate. Group Cy had thicker septum (median 0.85 vs 0.66 cm; P = .01) and longer crossclamp time (median 100.0 vs 67.5 minutes; P = .004). There were no difference in HSP-70i between groups at baseline (4.12 vs 3.44 relative optical density; P = .45) or subsequent ischemic time. Preoperative biventricular systolic function was reduced in patients with tetralogy compared with controls with further postoperative right ventricular impairment. Group Cy had higher troponin-I levels (median 16.5 vs 11.1 ng/mL; P = .04) and inotrope scores (14.0 vs 6.5; P = .05) but no differences in ventricular function, mixed venous oxygen saturation, oxygen extraction ratio, and lactate between groups. In group Cy, baseline HSP-70i correlated with better postoperative right ventricular function (rho = 0.80; P = .009), mixed venous oxygen saturation (rho = 0.68; P = .04), and oxygen extraction ratio (rho = -0.71; P = .03). These relationships were absent in group noCy. The association of HSP-70i expression with improved right ventricular function and systemic perfusion suggests an important cardioprotective effect of HSP-70i in cyanotic tetralogy of Fallot.

Activation of the stress protein response inhibits the STAT1 signalling pathway and iNOS function in alveolar macrophages: role of Hsp90 and Hsp70

Background and aimAlveolar fluid clearance is impaired by inducible nitric oxide synthase (iNOS)/nitric oxide (NO)-dependent mechanisms in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The activation of the stress...

Heat shock protein 90 inhibitors exhibit anti‐inflammatory response and differential cytoprotection pattern from oxidative stress‐ induced injury.

Geldanamycin have emerged as promising therapeutic agents in cancer. Accordingly, we tested the role of GA, 17AAG and radicicol in LPS‐ activated microglia. BV2 cells were treated with LPS with or without GA analogs. 24hr later, media and cell extracts were assayedfor NO and iNOS protein; in parallel injury was examined by viability assays. Pretreatment with GA and its analogs markedly prevent iNOS/NO accumulation elicited by LPS. Conversely, GA and its analogs prevented LPS ‐induced subsequent BV2 death. In addition, treatment of astrocytes with GA for 24hr induces increase of HSP70i expression. Moreover, we found that GA protect from oxidative stress induced by H2O2 in glial cells. To examine the mechanisms of delayed protection in LPS induced injury, cells were incubated with GA and analogs for 1 day, thereafter, cells were subjected to LPS for another 24hr. GA and to lesser extend 17AAG but not radicicol showed a partial blunting in pro‐inflammatory induction of iNOS/NO accumulation and display protection in the delayed window from LPS ‐induced BV2 cell death. Our data together, showed that GA and its analogs exhibit dual protection in brain resident cells: acutely by blocking the signaling that drive inflammatory response elicited by endotoxin or TLR3 agonist, and a delayed protection against oxidative stress. These data support usefulness of the HSP90 targeting to protect from neuro‐inflammatory disease.

Activation of the stress protein response inhibits the STAT1 signaling pathway and iNOS function in alveolar macrophages: role of Hsp90 and Hsp70

Alveolar fluid clearance is impaired by iNOS/NO‐dependent mechanisms in ALI/ARDS. We investigated whether activation of the stress protein response (SPR) by heat, or using an Hsp90 inhibitor inhibits the STAT1 signaling pathway, which is activated in ALI/ARDS, and thus inhibits iNOS/NO production. Inhibition of STAT1 activation after heat stress results in detergent insolubilization of the STAT1 protein and its proteasomal degradation which is reversed with the pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of heat‐induced protein denaturation. This early effect of SPR is the result of the disruption of the Hsp90 binding to the STAT1 protein and confirmed using an Hsp90 inhibitor. Recovery of STAT1 activation and iNOS synthesis occurs by 12 hours post‐SPR induction, however, NO production did not occur until 48 hours later. These results demonstrate a late effect of SPR activation involving the regulation of iNOS function by inducible Hsp70 (Hsp70i). Inhibiting Hsp70i expression after heat stress recovers iNOS function whereas overexpressing Hsp70i in the absence of heat stress inhibits iNOS function. These results represent novel mechanisms by which the activation of SPR inhibits STAT1 signaling in alveolar macrophages and highlight a potential clinical application for Hsp90 inhibitors in modulating NO signaling during the early phase of acute lung injury. [Research support: UCSF academic senate grant (MH); NIH GM62188 (JFP)]

Early Onset of Heat-Shock Response in Mouse Embryos Revealed by Quantification of Stress-Inducible hsp70i RNA

Heat shock response is fully established in mouse embryos at the blastocyst stage, but it is unclear when this response first arises during development. To shed light on this question, we used a single-tube method to quantify mRNA levels of the heat shock protein genes hsp70.1 and hsp70.3 ( hsp70i) in individual cleavage-stage embryos that had or had not been heat-shocked. While untreated, healthy embryos contained very low copy numbers of hsp70i RNA, heat shock rapidly induced the synthesis of hundreds of hsp70i transcripts per blastomere at both the 4-cell and the 8-cell stages. In addition, we performed hsp70i measurements in embryos that had not been heat-shocked but had been very slow in developing. Quantification of hsp70i RNA and genomic DNA copy numbers in these slow-growing embryos demonstrated the presence of two distinct populations. Some of the embryos contained considerable levels of hsp70i RNA, a finding consistent with the hypothesis of endogenous metabolic stress accompanied by cell cycle arrest and delayed development. Other slow-growing embryos contained no hsp70i RNA and fewer than expected hsp70i gene copies, suggesting the possibility of ongoing apoptosis. In conclusion, this study demonstrates that mouse embryos can activate hsp70i expression in response to sub-lethal levels of stress as early as at the 4-cell stage. Our results also indicate that quantification of hsp70i DNA and RNA copy numbers may provide a diagnostic tool for embryonic health.

Inducible 70 kDa heat shock protein does not protect spermatogenic cells from damage induced by cryptorchidism

Accumulation of inducible heat shock proteins (e.g. Hsp70i) during cellular stress confers thermotolerance, reduces the consequences of damage and facilitates cellular recovery, while abrogation of Hsp70i expression renders sensitivity to apoptosis. Testis translocation into abdominal cavity, which results in temperature elevation, does not induce expression of the Hsp70i proteins. Despite constitutive expression of testis-specific Hsp70 proteins, spermatocytes are very sensitive to damage at elevated temperatures. To test whether Hsp70i protein could protect testes from heat-induced damage, we have engineered transgenic mice that over-express this protein selectively in spermatocytes and spermatids. We demonstrate that the testes of cryptorchid transgenic mice, like those of wild-type mice, exhibit reduced weight and smaller sizes of their seminiferous tubules, disorganization of their germinal epithelium structures, appearance of multinucleated giant cells, and reduced populations of germ cells. The data show that constitutive expression of Hsp70i does not protect the seminiferous epithelium against cryptorchidism-induced damage.

In vivo gene delivery of HSP70i by adenovirus and adeno-associated virus preserves contractile function in mouse heart following ischemia-reperfusion

Inducible heat shock protein 70 (HSP70i) has been shown to exert a protective effect in hearts subjected to ischemia-reperfusion. Although studied in heat-shocked animals and in transgenic mice that constitutively overexpress the protein, the therapeutic application of the protein in the form of a viral vector-mediated HSP70i expression has not been widely examined. Accordingly, we have examined the effects of HSP70i delivered in vivo to the left ventricular free wall of the heart via viral gene therapy in mice. The affect of virally mediated HSP70i expression in preserving cardiac function following ischemia-reperfusion was examined after short-term expression (5-day adenovirus mediated) and long-term expression (8-mo adeno-associated virus mediated) in mice by subjecting ex vivo Langendorff perfused hearts to a regime of ischemia-reperfusion. Both vectors were capable of increasing HSP70i expression in the heart, and neither vector had any effect on cardiac function during aerobic (preischemic) perfusion when compared with corresponding controls. In contrast, both adenovirus-mediated and adeno-associated virus-mediated expression of HSP70i improved the contractile recovery of the heart after 120 min of reperfusion following ischemia. This study demonstrates the feasibility of using both short- and long-term expression of virally mediated HSP70i as a therapeutic intervention against cardiac ischemia-reperfusion injury.

Heat Shock Protein 25 or Inducible Heat Shock Protein 70 Activates Heat Shock Factor 1: DEPHOSPHORYLATION ON SERINE 307 THROUGH INHIBITION OF ERK1/2 PHOSPHORYLATION

The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.

Increased hepatotoxicity of acetaminophen in Hsp70i knockout mice

The effect of the inducible forms of 70 kDa heat shock protein (Hsp70i) on acetaminophen (APAP) hepatotoxicity was assessed in an Hsp70i knockout mouse model. Absence of the Hsp70i protein in liver was verified by monitoring Hsp levels in knockout and control mice after heat stress (41.5 degrees C water bath immersion for 30 min). Hsp70i knockout mice were more susceptible to APAP-induced hepatotoxicity than controls, as indicated by elevated serum alanine aminotransferase activities 24 and 48 h after the APAP dose. Increased APAP hepatotoxicity in knockout mice was verified by morphological evaluation of liver sections. The difference in toxic response to APAP between knockout and control strain mice could not be attributed to differences in APAP bioactivation, assessed by measurement of CYP2E1 and glutathione S-transferase activities, hepatic nonprotein sulfhydryl content, or covalent binding of reactive APAP metabolites to proteins. Pretreatment with transient hyperthermia to produce a general upregulation of Hsps resulted in decreased APAP hepatotoxicity in both the knockout and control strains. Among thermally-pretreated mice, hepatotoxicity of APAP was greater in the knockouts compared with the control strain. These observations suggest that increased Hsp70i expression in response to APAP acts to limit the extent of tissue injury. Results further suggest that other factors related to heat stress can also contribute to protection against APAP toxicity.

Laser zona drilling does not induce hsp70i transcription in blastomeres of eight-cell mouse embryos

To assess whether zona drilling with a 1,480-nm laser induces heat shock in eight-cell embryos, we measured hsp70i RNA levels in sets of single blastomeres isolated after laser treatment of mouse embryos that had or had not been heated at 43 degrees C. Unlike heating, laser zona drilling did not stimulate hsp70i expression, even in the blastomeres closest to the laser beam, corroborating the safety of this procedure for assisted reproduction.

Spermatocyte-specific expression of constitutively active heat shock factor 1 induces HSP70i-resistant apoptosis in male germ cells

Spermatocytes, the most sensitive male germ cells to heat-induced apoptosis, do not respond to hyperthermia by inducing heat shock proteins (HSPs), including HSP70i, which has been previously shown to confer resistance to apoptosis in somatic cells. To dissect the mechanism of heat-induced apoptosis and to determine if we could protect spermatocytes by expressing HSP70i, we engineered transgenic mice that express in spermatocytes constitutively active heat shock transcription factor (HSF)1. Such HSF1 expression did not lead to transcription of inducible Hsp70 genes, but instead induced caspase-dependent apoptosis that mimicked heat shock-induced death of spermatogenic cells. Both mitochondria-dependent and death receptor-dependent pathways appear to be involved in such HSF1-induced apoptosis: the levels of Bcl-2 family proteins became increased, p53 protein accumulated and expression levels of caspase-8 and death-receptor-interacting proteins (including Fas-associated death domain protein and TNF receptor associated death domain protein) became elevated. Surprisingly, the constitutive spermatocyte-specific expression of HSP70i in double-transgenic males did not protect against such HSF1-induced apoptosis.

Heat shock protein 70 expression, keratin phosphorylation and Mallory body formation in hepatocytes from griseofulvin-intoxicated mice

BackgroundKeratins are members of the intermediate filaments (IFs) proteins, which constitute one of the three major cytoskeletal protein families. In hepatocytes, keratin 8 and 18 (K8/18) are believed to play a protective role against mechanical and toxic stress. Post-translational modifications such as phosphorylation and glycosylation are thought to modulate K8/18 functions. Treatment of mouse with a diet containing griseofulvin (GF) induces, in hepatocytes, modifications in organization, expression and phosphorylation of K8/18 IFs and leads, on the long term, to the formation of K8/18 containing aggregates morphologically and biochemically identical to Mallory bodies present in a number of human liver diseases. The aim of the present study was to investigate the relationship between the level and localization of the stress inducible heat shock protein 70 kDa (HSP70i) and the level and localization of K8/18 phosphorylation in the liver of GF-intoxicated mice. The role of these processes in Mallory body formation was studied, too. The experiment was carried out parallely on two different mouse strains, C3H and FVB/n.ResultsGF-treatment induced an increase in HSP70i expression and K8 phosphorylation on serines 79 (K8 S79), 436 (K8 S436), and K18 phosphorylation on serine 33 (K18 S33) as determined by Western blotting. Using immunofluorescence staining, we showed that after treatment, HSP70i was present in all hepatocytes. However, phosphorylated K8 S79 (K8 pS79) and K8 S436 (K8 pS436) were observed only in groups of hepatocytes or in isolated hepatocytes. K18 pS33 was increased in all hepatocytes. HSP70i colocalized with MBs containing phosphorylated K8/18. Phophorylation of K8 S79 was observed in C3H mice MBs but was not present in FVB/n MBs.ConclusionsOur results indicate that GF intoxication represents a stress condition affecting all hepatocytes, whereas induction of K8/18 phosphorylation is not occurring in every hepatocyte. We conclude that, in vivo, there is no direct relationship between GF-induced stress and K8/18 phosphorylation on the studied sites. The K8/18 phosphorylation pattern indicates that different cell signaling pathways are activated in subpopulations of hepatocytes. Moreover, our results demonstrate that, in distinct genetic backgrounds, the induction of K8/18 phosphorylation can be different.