Increases in HSF1 translocation and synthesis in human epidermoid A-431 Cells: role of protein kinase C and [Ca2+]i

Xuan Z. Ding, Robert Christian Smallridge, Smallridge, Richard J. Galloway, G. Juliann, Kiang

Research output: Contribution to journalArticle

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Abstract

Background: It is known that heat shock increases both heat shock protein 70 kd (HSP-70), mRNA synthesis, and intracellular cytosolic free calcium concentration ([Ca2+]i). The latter enhances the heat inducible form of HSP-70 production by increasing the complex formation between heat shock transcriptional factor (HSF) and heat shock elements (HSE). In this study, we investigated the effect of agonists (PMA; ionomycin) and antagonists (BAPTA; staurosporine) of protein kinase C (PKC), and calcium channel on translocation and synthesis of HSF1, and activation of HSP-70 gene in human epidermoid A-431 cells. Methods: Cells were incubated with poly 12-myristate 13-acetate (PMA) or ionomycin at different concentrations for various periods of time. Messenger RNAs of HSF and HSP70 were measured with RT-PCR. The HSP-70 protein was determined with Western blots, and HSF protein was measured by gel mobility retardation assay. Results: Significant increases in HSF binding to [32P]labeled HSE were found at 30 minutes in nuclear extract and at 4 hours in both nuclear and cytosol extracts. The PMA- and ionomycin-induced increases in HSF were in a concentration-dependent manner with a maximal increase at 10-6 mol/L of each drug. Meanwhile, the mRNAs encoded for HSF1 and HSP-70, but not HSF2, were significantly increased and reached the maximum at 1 hour after the treatment. The PMA increased [Ca2+]i by 92% because of Ca2+ influx. The increases in mRNA of HSF1 and HSP-70 induced by treatment with 1 umol/L PMA were completely blocked by preincubating cells with either 2 umol/L staurosporine in the presence of extracellular Ca2+ or 100 umol/L BAPTA-am in absence of extracellular Ca2+. Like PMA, the increases induced by ionomycin were also inhibited by 100 pmol/L BAPTA-am in absence of extracellular Ca2+. Furthermore, Western blots show that 1 umol/L PMA or ionomycin induced maximal increase in HSP-70 after 7hours of continuous incubation with either agent. When cells were simultaneously treated with 1 umol/L PMA and ionomycin together for 1 hour, the increase in HSP-70 and HSF1 mRNAs reached a greater level than the level stimulated by either drug alone. Conclusions: These results indicate that both PMA and ionomycin stimulate HSF1, but not HSF2, translocation and synthesis leading to the HSP-70 expression and that their effects are Ca2+-dependent.

Original languageEnglish (US)
Pages (from-to)144-153
Number of pages10
JournalJournal of Investigative Medicine
Volume44
Issue number4
StatePublished - 1996
Externally publishedYes

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HSP70 Heat-Shock Proteins
Myristic Acid
Ionomycin
Protein Kinase C
Acetates
Hot Temperature
Shock
Messenger RNA
Staurosporine
Western Blotting
Electrophoretic Mobility Shift Assay
Calcium Channels
Pharmaceutical Preparations
Cytosol
Assays
Proteins
Genes
Gels
Chemical activation
Calcium

Keywords

  • Calcium
  • Epithelia
  • Heat shock factor
  • Heat shock proteins
  • Protein kinase c

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Increases in HSF1 translocation and synthesis in human epidermoid A-431 Cells : role of protein kinase C and [Ca2+]i. / Ding, Xuan Z.; Smallridge, Robert Christian; Smallridge; Galloway, Richard J.; Juliann, G.; Kiang.

In: Journal of Investigative Medicine, Vol. 44, No. 4, 1996, p. 144-153.

Research output: Contribution to journalArticle

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abstract = "Background: It is known that heat shock increases both heat shock protein 70 kd (HSP-70), mRNA synthesis, and intracellular cytosolic free calcium concentration ([Ca2+]i). The latter enhances the heat inducible form of HSP-70 production by increasing the complex formation between heat shock transcriptional factor (HSF) and heat shock elements (HSE). In this study, we investigated the effect of agonists (PMA; ionomycin) and antagonists (BAPTA; staurosporine) of protein kinase C (PKC), and calcium channel on translocation and synthesis of HSF1, and activation of HSP-70 gene in human epidermoid A-431 cells. Methods: Cells were incubated with poly 12-myristate 13-acetate (PMA) or ionomycin at different concentrations for various periods of time. Messenger RNAs of HSF and HSP70 were measured with RT-PCR. The HSP-70 protein was determined with Western blots, and HSF protein was measured by gel mobility retardation assay. Results: Significant increases in HSF binding to [32P]labeled HSE were found at 30 minutes in nuclear extract and at 4 hours in both nuclear and cytosol extracts. The PMA- and ionomycin-induced increases in HSF were in a concentration-dependent manner with a maximal increase at 10-6 mol/L of each drug. Meanwhile, the mRNAs encoded for HSF1 and HSP-70, but not HSF2, were significantly increased and reached the maximum at 1 hour after the treatment. The PMA increased [Ca2+]i by 92{\%} because of Ca2+ influx. The increases in mRNA of HSF1 and HSP-70 induced by treatment with 1 umol/L PMA were completely blocked by preincubating cells with either 2 umol/L staurosporine in the presence of extracellular Ca2+ or 100 umol/L BAPTA-am in absence of extracellular Ca2+. Like PMA, the increases induced by ionomycin were also inhibited by 100 pmol/L BAPTA-am in absence of extracellular Ca2+. Furthermore, Western blots show that 1 umol/L PMA or ionomycin induced maximal increase in HSP-70 after 7hours of continuous incubation with either agent. When cells were simultaneously treated with 1 umol/L PMA and ionomycin together for 1 hour, the increase in HSP-70 and HSF1 mRNAs reached a greater level than the level stimulated by either drug alone. Conclusions: These results indicate that both PMA and ionomycin stimulate HSF1, but not HSF2, translocation and synthesis leading to the HSP-70 expression and that their effects are Ca2+-dependent.",
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T1 - Increases in HSF1 translocation and synthesis in human epidermoid A-431 Cells

T2 - role of protein kinase C and [Ca2+]i

AU - Ding, Xuan Z.

AU - Smallridge, Robert Christian

AU - Smallridge,

AU - Galloway, Richard J.

AU - Juliann, G.

AU - Kiang,

PY - 1996

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N2 - Background: It is known that heat shock increases both heat shock protein 70 kd (HSP-70), mRNA synthesis, and intracellular cytosolic free calcium concentration ([Ca2+]i). The latter enhances the heat inducible form of HSP-70 production by increasing the complex formation between heat shock transcriptional factor (HSF) and heat shock elements (HSE). In this study, we investigated the effect of agonists (PMA; ionomycin) and antagonists (BAPTA; staurosporine) of protein kinase C (PKC), and calcium channel on translocation and synthesis of HSF1, and activation of HSP-70 gene in human epidermoid A-431 cells. Methods: Cells were incubated with poly 12-myristate 13-acetate (PMA) or ionomycin at different concentrations for various periods of time. Messenger RNAs of HSF and HSP70 were measured with RT-PCR. The HSP-70 protein was determined with Western blots, and HSF protein was measured by gel mobility retardation assay. Results: Significant increases in HSF binding to [32P]labeled HSE were found at 30 minutes in nuclear extract and at 4 hours in both nuclear and cytosol extracts. The PMA- and ionomycin-induced increases in HSF were in a concentration-dependent manner with a maximal increase at 10-6 mol/L of each drug. Meanwhile, the mRNAs encoded for HSF1 and HSP-70, but not HSF2, were significantly increased and reached the maximum at 1 hour after the treatment. The PMA increased [Ca2+]i by 92% because of Ca2+ influx. The increases in mRNA of HSF1 and HSP-70 induced by treatment with 1 umol/L PMA were completely blocked by preincubating cells with either 2 umol/L staurosporine in the presence of extracellular Ca2+ or 100 umol/L BAPTA-am in absence of extracellular Ca2+. Like PMA, the increases induced by ionomycin were also inhibited by 100 pmol/L BAPTA-am in absence of extracellular Ca2+. Furthermore, Western blots show that 1 umol/L PMA or ionomycin induced maximal increase in HSP-70 after 7hours of continuous incubation with either agent. When cells were simultaneously treated with 1 umol/L PMA and ionomycin together for 1 hour, the increase in HSP-70 and HSF1 mRNAs reached a greater level than the level stimulated by either drug alone. Conclusions: These results indicate that both PMA and ionomycin stimulate HSF1, but not HSF2, translocation and synthesis leading to the HSP-70 expression and that their effects are Ca2+-dependent.

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KW - Epithelia

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KW - Protein kinase c

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