Activation of heme oxygenase and heat shock protein 70 genes by stress in human hepatoma cells

Effects of various stresses were examined on the accumulation of mRNA for microsomal heme oxygenase and a heat shock protein, hsp70, in three human hepatoma cell lines. By heat shock, hsp70 mRNA was induced in all three hepatoma lines, Hep G2, Hep 3B and Hep G2f, while heme oxygenase mRNA was increased only in Hep 3B. Time-courses of the heat shock induction of both mRNAs in Hep 3B were similar. Arsenite caused induction of both mRNAs in all three cell lines, while cadmium increased them in Hep G2 and Hep 3B, but not in Hep G2f cells. These findings suggest that, although both hsp70 and heme oxygenase are heat shock proteins, the mode of induction of mRNAs for these proteins is different.     

Adenovirus early gene products may control viral mRNA accumulation and translation in vivo

The mechanisms controlling early adenovirus gene expression in vivo have been studied using inhibitors of protein synthesis. When inhibitors were added shortly before or at the onset of infection, viral mRNA from all early regions was transcribed, spliced and accumulated over a 7 hr period. After longer pretreatment, accumulation of several early mRNAs were suppressed. Addition of inhibitors 1 hr after infection enhanced the accumulation of viral mRNA in the cytoplasm. Translation of early mRNA selected on adenovirus DNA in a cell-free system reflected the amount of viral mRNA present. A viral coded product may therefore control accumulation of viral mRNA.A different pattern emerged when inhibitors of protein synthesis were removed at 5 hr postinfection and cells were pulse-labeled in vivo. If inhibitors were introduced at or before infection, early viral proteins were synthesized only after a lag of 1–3 hr. However, if treatment was introduced 1 hr post-infection, reversion of the protein synthesis block was instantaneous. It appears that protein synthesis inhibitors reveal an in vivo translational block for viral mRNA. This block could be overcome by preinfection with a related virus. Furthermore, no block was observed in a virus-transformed human embryonic kidney cell line (293) which expresses early region 1 of the viral genome. Viral gene product(s) encoded in early region 1 may control translation of early adenovirus messenger RNA in vivo.     

Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport.

Late in adenovirus infection, large amounts of viral mRNA accumulate while cell mRNA transport and translation decrease. Viruses deleted in the E1B region of type 5 adenovirus do not produce the same outcome: (i) viral mRNA synthesis by the mutants is normal, delivery to the cytoplasm is 50 to 75% of normal, but steady-state levels of viral mRNA are decreased 10-fold; (ii) cell mRNA synthesis and transport continue normally in the mutant virus-infected cell; and (iii) translation of preexisting cell mRNA which is disrupted in wild-type infection remains normal in mutant-virus-infected cells. Thus E1B proteins are required for accumulation of virus mRNA and for induction of the failure of host cell mRNA transport and translation. If a single function is involved, by inference the transport and some aspect of translation of mRNAs could be linked.     

Protein ligands mediate the CRM1-dependent export of HuR in response to heat shock

AU-rich elements (AREs) located in the 3' UTRs of the messenger RNAs (mRNAs) of many mammalian early response genes promote rapid mRNA turnover. HuR, an RRM-containing RNA-binding protein, specifically interacts with AREs, stabilizing these mRNAs. HuR is primarily nucleoplasmic, but shuttles between the nucleus and the cytoplasm via a domain called HNS located between RRM2 and RRM3. We recently showed that HuR interacts with two protein ligands, pp32 and APRIL, which are also shuttling proteins, but rely on NES domains recognized by CRM1 for export. Here we show that heat shock induces increased association of HuR with pp32 and APRIL through protein-protein interactions and that these ligands partially colocalize with HuR in cytoplasmic foci. HuR associations with the hnRNP complex also increase, but through RNA links. CRM1 coimmunoprecipitates with HuR only after heat shock, and nuclear export of HuR becomes sensitive to leptomycin B, an inhibitor of CRM1. Export after heat shock requires the same domains of HuR (HNS and RRM3) that are essential for binding pp32 and APRIL. In situ hybridization and coimmunoprecipitation experiments show that LMB treatment blocks both hsp70 mRNA nuclear export and its cytoplasmic interaction with HuR after heat shock. Together, our results argue that upon heat shock, HuR switches its export pathway to that of its ligands pp32 and APRIL, which involves the nuclear export factor CRM1. HuR and its ligands may be instrumental in the nuclear export of heat-shock mRNAs.     

Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader.

One of the effects of a temperature increase above 35 degrees C on Drosophila melanogaster is a rapid switch in selectivity of the translational apparatus. Protein synthesis from normal, but not from heat shock, mRNA is much reduced. Efficient translation at high temperature might be a result of the primary sequence of heat shock genes. Alternatively a mRNA modification mechanism, altered as a consequence of heat shock, might allow for efficient high temperature translation of any mRNA synthesized during a heat shock. The gene for alcohol dehydrogenase (Adh) was fused to the controlling elements of a heat shock protein 70 (hsp70) gene. Authentic Adh mRNA, synthesized from this fusion gene at elevated temperatures was not translated during heat shock. A second Adh fusion gene in which the mRNA synthesized contained the first 95 nucleotides of the Hsp70 non-translated leader sequence gave rise, at high temperature, to mRNA which was translated during the heat shock. Thus, the signal(s) in the mRNAs controlling translation efficiency at heat shock temperatures is encoded within the heat shock genes.     

Regulation of HSP70 synthesis by messenger RNA degradation.

When Drosophila cells are heat shocked, hsp70 messenger RNA (mRNA) is stable and is translated at high efficiencies. During recovery from heat shock, hsp70 synthesis is repressed and its messenger RNA (mRNA) is degraded in a highly regulated fashion. Dramatic differences in the timing of repression and degradation are observed after heat treatments of different severities. The 3' untranslated region (UTR) of the hsp70 mRNA was sufficient to transfer this regulated degradation to heterologous mRNAs. Altering the translational efficiency of the message or changing its natural translation-termination site did not alter its pattern of regulation, although in some cases it changed the absolute rate of degradation. We have previously shown that hsp70 mRNA is very unstable when it is expressed at normal growth temperatures (from a metallothionein promoter). We report here that the 3' untranslated region of the hsp70 mRNA is responsible for this instability as well. We postulate that a mechanism for degrading hsp70 mRNA pre-exists in Drosophila cells, that it is inactivated by heat shock and that it is the reactivation of this mechanism that is responsible for hsp70 repression during recovery. This degradation system may be the same as that used by other unstable mRNAs.     

Differential distribution of the adenovirus E1A proteins and colocalization of E1A with the 70-kilodalton cellular heat shock protein in infected cells.

Five distinct localization patterns were observed for the adenovirus E1A proteins in the nuclei of infected HeLa cells: diffuse, reticular, nucleolar, punctate, and peripheral. The variable distribution of E1A was correlated with the time postinfection and the cell cycle stage of the host cell at the time of infection. All staining patterns, with the exception of peripheral E1A localization, were associated with the early phase of infection since only the diffuse, reticular, nucleolar, and punctate staining patterns were observed in the presence of hydroxyurea. Because the E1A proteins (12S and 13S) stimulate the expression of the cellular heat shock 70-kilodalton protein (hsp70), we examined the intracellular distribution of hsp70 in the adenovirus-infected cells. Whereas hsp70 was predominantly cytoplasmic in the cells before infection, after adenovirus infection most of the protein was now found within the nucleus. Specifically, hsp70 was found within the nucleoli as well as exhibiting reticular, diffuse, and punctate nuclear staining patterns, analogous to those observed for the E1A proteins. Double-label indirect immunofluorescence of E1A and hsp70 in infected cells demonstrated a colocalization of these proteins in the nucleus. Translocation of hsp70 to the nucleus was dependent upon both adenovirus infection and expression of the E1A proteins. The localization of hsp70 was unaltered by infection with an E1A 9S cDNA virus which does not synthesize a functional E1A gene product. Moreover, the discrete nuclear localization patterns of E1A and the colocalization of E1A with hsp70 were not observed in adenovirus-transformed 293 cells which constitutively express E1A and E1B. E1A displayed exclusively diffuse nuclear staining in 293 cells; however, localization of E1A into the discrete nuclear patterns occurred after adenovirus infection of 293 cells. Immunoprecipitation of labeled infected-cell extracts with a monoclonal antibody directed against the E1A proteins resulted in precipitation of small amounts of hsp70 along with E1A. These data indicate that the adenovirus E1A proteins colocalize with, and possibly form a physical complex with, cellular hsp70 in infected cells. The relevance of this association, with respect to the function of these proteins during infection and the association of other oncoproteins with hsp70, is discussed.     

Hydrogen peroxide induces heat shock protein and proto-oncogene mRNA accumulation in Xenopus laevis A6 kidney epithelial cells

In this study, we examined the effect of hydrogen peroxide on the accumulation of various mRNAs encoding heat shock proteins (hsps) and proto-oncogenes in Xenopus A6 kidney epithelial cells. Hydrogen peroxide treatment enhanced the accumulation of hsp90, hsp70, hsp30, c-jun, c-fos, and actin mRNAs with distinct temporal patterns. Although hsp70, c-fos, and c-jun mRNA levels peaked at 1-2 h before declining, hsp30 and hsp90 mRNA levels were maximal at 4-6 h. Other mRNAs, including heat shock cognate hsc70, immunoglobulin binding protein, and ribosomal L8, were unaffected. Treatment of kidney cells with a combination of mild heat shock plus hydrogen peroxide resulted in a synergistic increase in the relative levels of both hsp70 and hsp30 mRNA, but not hsp90, c-fos, c-jun, or actin. This study suggests that analysis of hsp and proto-oncogene mRNA levels may be of value as molecular biomarkers of oxidative stress associated with various disease states and nephrotoxicity in kidney.     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.