Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1997 Jan 1;99(1):136–143. doi: 10.1172/JCI119124

Heat shock factor-1 protein in heat shock factor-1 gene-transfected human epidermoid A431 cells requires phosphorylation before inducing heat shock protein-70 production.

X Z Ding 1, G C Tsokos 1, J G Kiang 1
PMCID: PMC507777  PMID: 9011567

Abstract

Heat shock factor-1 (HSF1) is a transcriptional factor that binds to heat shock elements located on the promoter region of heat shock protein genes. The purpose of this study was to further investigate the regulation of the expression of the heat shock protein-70 (HSP-70) gene. The HSF1 gene was inserted into pCDNA3 plasmid and then transfected into human epidermoid A431 cells using the CaOP3 method. Control cells were transfected with vector alone. Expression of HSP-70, HSF1, and HSF2 genes and protein were determined. We found a significant increase in the expression of the HSF1 gene, but not HSP-70 and HSF2 genes, in the HSF1 gene-transfected cells. The amount of HSF1-heat shock element complex was significantly increased in both the nucleus and cytosol in HSF1 gene-transfected cells, indicating increased synthesis of HSF1. The amount of HSP-72 in these cells did not change. Therefore, overexpression of HSF1 protein failed to initiate transcription of the HSP-70 gene. Subsequently, we treated the cells with 1 microM PMA (a protein kinase C stimulator), and HSP-70 mRNA and protein were measured at 1 or 4 h of the treatment, respectively. The levels of both HSP-70 mRNA and HSP-72 protein were significantly increased in nontransfected and transfected cells; the levels of HSP-72 in HSF1 gene-transfected cells were greater than that found in the vector-transfected cells. The PMA-induced increase in HSP-72 protein peaked 8 h after treatment with PMA and returned to baseline levels at 72 h. This increase was blocked by a PKC inhibitor, staurosporine. After treatment with PMA, HSF1 translocated quickly from cytosol to nucleus. The results suggest that phosphorylation of newly synthesized HSF1 and possibly of other factors are necessary for the induction of HSP-72. Activation of PKC can cause phosphorylation of HSF1, which leads to an enhanced but transient increase in HSP-70 production.

Full Text

The Full Text of this article is available as a PDF (274.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abravaya K., Phillips B., Morimoto R. I. Heat shock-induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting. Mol Cell Biol. 1991 Jan;11(1):586–592. doi: 10.1128/mcb.11.1.586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbe M. F., Tytell M., Gower D. J., Welch W. J. Hyperthermia protects against light damage in the rat retina. Science. 1988 Sep 30;241(4874):1817–1820. doi: 10.1126/science.3175623. [DOI] [PubMed] [Google Scholar]
  3. Blake M. J., Udelsman R., Feulner G. J., Norton D. D., Holbrook N. J. Stress-induced heat shock protein 70 expression in adrenal cortex: an adrenocorticotropic hormone-sensitive, age-dependent response. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9873–9877. doi: 10.1073/pnas.88.21.9873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Currie R. W., Karmazyn M., Kloc M., Mailer K. Heat-shock response is associated with enhanced postischemic ventricular recovery. Circ Res. 1988 Sep;63(3):543–549. doi: 10.1161/01.res.63.3.543. [DOI] [PubMed] [Google Scholar]
  5. Dewji N. N., Do C., Bayney R. M. Transcriptional activation of Alzheimer's beta-amyloid precursor protein gene by stress. Brain Res Mol Brain Res. 1995 Nov;33(2):245–253. doi: 10.1016/0169-328x(95)00131-b. [DOI] [PubMed] [Google Scholar]
  6. Ding X. Z., Smallridge R. C., Galloway R. J., Kiang J. G. Increases in HSF1 translocation and synthesis in human epidermoid A-431 cells: role of protein kinase C and [Ca2+]i. J Investig Med. 1996 Apr;44(4):144–153. [PubMed] [Google Scholar]
  7. Ding X. Z., Smallridge R. C., Galloway R. J., Kiang J. G. Rapid assay of HSF1 and HSF2 gene expression by RT-PCR. Mol Cell Biochem. 1996 May 24;158(2):189–192. doi: 10.1007/BF00225845. [DOI] [PubMed] [Google Scholar]
  8. Fargnoli J., Kunisada T., Fornace A. J., Jr, Schneider E. L., Holbrook N. J. Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats. Proc Natl Acad Sci U S A. 1990 Jan;87(2):846–850. doi: 10.1073/pnas.87.2.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hansen L. K., Houchins J. P., O'Leary J. J. Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes. Exp Cell Res. 1991 Feb;192(2):587–596. doi: 10.1016/0014-4827(91)90080-e. [DOI] [PubMed] [Google Scholar]
  10. Hosokawa N., Hirayoshi K., Kudo H., Takechi H., Aoike A., Kawai K., Nagata K. Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. Mol Cell Biol. 1992 Aug;12(8):3490–3498. doi: 10.1128/mcb.12.8.3490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Høj A., Jakobsen B. K. A short element required for turning off heat shock transcription factor: evidence that phosphorylation enhances deactivation. EMBO J. 1994 Jun 1;13(11):2617–2624. doi: 10.1002/j.1460-2075.1994.tb06552.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jurivich D. A., Sistonen L., Sarge K. D., Morimoto R. I. Arachidonate is a potent modulator of human heat shock gene transcription. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2280–2284. doi: 10.1073/pnas.91.6.2280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaufmann S. H. Heat shock proteins and the immune response. Immunol Today. 1990 Apr;11(4):129–136. doi: 10.1016/0167-5699(90)90050-j. [DOI] [PubMed] [Google Scholar]
  14. Kiang J. G., Carr F. E., Burns M. R., McClain D. E. HSP-72 synthesis is promoted by increase in [Ca2+]i or activation of G proteins but not pHi or cAMP. Am J Physiol. 1994 Jul;267(1 Pt 1):C104–C114. doi: 10.1152/ajpcell.1994.267.1.C104. [DOI] [PubMed] [Google Scholar]
  15. Kiang J. G., Koenig M. L. Characterization of intracellular calcium pools and their desensitization in thermotolerant human A-431 cells. J Investig Med. 1996 Aug;44(6):352–361. [PubMed] [Google Scholar]
  16. Kiang J. G., Koenig M. L., Smallridge R. C. Heat shock increases cytosolic free Ca2+ concentration via Na(+)-Ca2+ exchange in human epidermoid A 431 cells. Am J Physiol. 1992 Jul;263(1 Pt 1):C30–C38. doi: 10.1152/ajpcell.1992.263.1.C30. [DOI] [PubMed] [Google Scholar]
  17. Kiang J. G., Wang X. D., Ding X. Z., Gist I. D., Smallridge R. C. Heat shock inhibits the hypoxia-induced effects on iodide uptake and signal transduction and enhances cell survival in rat thyroid FRTL-5 cells. Thyroid. 1996 Oct;6(5):475–483. doi: 10.1089/thy.1996.6.475. [DOI] [PubMed] [Google Scholar]
  18. Kim D., Ouyang H., Li G. C. Heat shock protein hsp70 accelerates the recovery of heat-shocked mammalian cells through its modulation of heat shock transcription factor HSF1. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2126–2130. doi: 10.1073/pnas.92.6.2126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kroeger P. E., Sarge K. D., Morimoto R. I. Mouse heat shock transcription factors 1 and 2 prefer a trimeric binding site but interact differently with the HSP70 heat shock element. Mol Cell Biol. 1993 Jun;13(6):3370–3383. doi: 10.1128/mcb.13.6.3370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mestril R., Chi S. H., Sayen M. R., Dillmann W. H. Isolation of a novel inducible rat heat-shock protein (HSP70) gene and its expression during ischaemia/hypoxia and heat shock. Biochem J. 1994 Mar 15;298(Pt 3):561–569. doi: 10.1042/bj2980561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mivechi N. F., Giaccia A. J. Mitogen-activated protein kinase acts as a negative regulator of the heat shock response in NIH3T3 cells. Cancer Res. 1995 Dec 1;55(23):5512–5519. [PubMed] [Google Scholar]
  22. Mosser D. D., Duchaine J., Massie B. The DNA-binding activity of the human heat shock transcription factor is regulated in vivo by hsp70. Mol Cell Biol. 1993 Sep;13(9):5427–5438. doi: 10.1128/mcb.13.9.5427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mosser D. D., Kotzbauer P. T., Sarge K. D., Morimoto R. I. In vitro activation of heat shock transcription factor DNA-binding by calcium and biochemical conditions that affect protein conformation. Proc Natl Acad Sci U S A. 1990 May;87(10):3748–3752. doi: 10.1073/pnas.87.10.3748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nakamura K., Rokutan K., Marui N., Aoike A., Kawai K. Induction of heat shock proteins and their implication in protection against ethanol-induced damage in cultured guinea pig gastric mucosal cells. Gastroenterology. 1991 Jul;101(1):161–166. doi: 10.1016/0016-5085(91)90473-x. [DOI] [PubMed] [Google Scholar]
  25. Newton E. M., Knauf U., Green M., Kingston R. E. The regulatory domain of human heat shock factor 1 is sufficient to sense heat stress. Mol Cell Biol. 1996 Mar;16(3):839–846. doi: 10.1128/mcb.16.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Perdrizet G. A., Kaneko H., Buckley T. M., Fishman M. A., Schweizer R. T. Heat shock protects pig kidneys against warm ischemic injury. Transplant Proc. 1990 Apr;22(2):460–461. [PubMed] [Google Scholar]
  27. Polla B. S., Healy A. M., Wojno W. C., Krane S. M. Hormone 1 alpha,25-dihydroxyvitamin D3 modulates heat shock response in monocytes. Am J Physiol. 1987 Jun;252(6 Pt 1):C640–C649. doi: 10.1152/ajpcell.1987.252.6.C640. [DOI] [PubMed] [Google Scholar]
  28. Price B. D., Calderwood S. K. Ca2+ is essential for multistep activation of the heat shock factor in permeabilized cells. Mol Cell Biol. 1991 Jun;11(6):3365–3368. doi: 10.1128/mcb.11.6.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rabindran S. K., Giorgi G., Clos J., Wu C. Molecular cloning and expression of a human heat shock factor, HSF1. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6906–6910. doi: 10.1073/pnas.88.16.6906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sarge K. D., Murphy S. P., Morimoto R. I. Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress. Mol Cell Biol. 1993 Mar;13(3):1392–1407. doi: 10.1128/mcb.13.3.1392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schlesinger M. J. Heat shock proteins. J Biol Chem. 1990 Jul 25;265(21):12111–12114. [PubMed] [Google Scholar]
  32. Sorger P. K. Heat shock factor and the heat shock response. Cell. 1991 May 3;65(3):363–366. doi: 10.1016/0092-8674(91)90452-5. [DOI] [PubMed] [Google Scholar]
  33. Stojadinovic A., Kiang J., Smallridge R., Galloway R., Shea-Donohue T. Induction of heat-shock protein 72 protects against ischemia/reperfusion in rat small intestine. Gastroenterology. 1995 Aug;109(2):505–515. doi: 10.1016/0016-5085(95)90339-9. [DOI] [PubMed] [Google Scholar]
  34. Sugawara S., Takeda K., Lee A., Dennert G. Suppression of stress protein GRP78 induction in tumor B/C10ME eliminates resistance to cell mediated cytotoxicity. Cancer Res. 1993 Dec 15;53(24):6001–6005. [PubMed] [Google Scholar]
  35. Van Why S. K., Mann A. S., Thulin G., Zhu X. H., Kashgarian M., Siegel N. J. Activation of heat-shock transcription factor by graded reductions in renal ATP, in vivo, in the rat. J Clin Invest. 1994 Oct;94(4):1518–1523. doi: 10.1172/JCI117492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Villar J., Ribeiro S. P., Mullen J. B., Kuliszewski M., Post M., Slutsky A. S. Induction of the heat shock response reduces mortality rate and organ damage in a sepsis-induced acute lung injury model. Crit Care Med. 1994 Jun;22(6):914–921. [PubMed] [Google Scholar]
  37. Yamamoto N., Smith M. W., Maki A., Berezesky I. K., Trump B. F. Role of cytosolic Ca2+ and protein kinases in the induction of the hsp70 gene. Kidney Int. 1994 Apr;45(4):1093–1104. doi: 10.1038/ki.1994.146. [DOI] [PubMed] [Google Scholar]
  38. Zimarino V., Tsai C., Wu C. Complex modes of heat shock factor activation. Mol Cell Biol. 1990 Feb;10(2):752–759. doi: 10.1128/mcb.10.2.752. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES