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Human Keratinocytes Respond to Osmotic Stress by p38 Map Kinase Regulated Induction of HSP70 and HSP27

2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês

10.1046/j.0022-202x.2001.01553.x

1523-1747

Marjan Garmyn, Annemie Pupe, T. Mammone, David Gan, L. Declercq, Daniel Maes,

Insect and Pesticide Research

Human skin is exposed to an environment that varies in humidity from 100 to 0%, leading to seasonal variations in the condition of the skin. Exposure to a low humidity environment creates an osmotic gradient across the stratum corneum, which is known to modulate cutaneous barrier function. Heat shock proteins protect against stress-induced destabilization of proteins. We investigated whether osmotic shock (sorbitol) induced a heat shock protein response in normal human keratinocytes, and used heat shock as a positive control. Both heat shock and osmotic stress (200 and 300 mM sorbitol) clearly induced heat shock proteins 70 and 27 mRNA levels. The induction of heat shock protein 70 mRNA levels by osmotic stress peaked at 16 h and persisted until 24 h, whereas upregulation of heat shock protein 70 mRNA levels by heat peaked at 2 h and returned to baseline levels by 6 h. Sorbitol also increased heat shock protein 70 levels in a concentration-dependent manner. The kinetics of heat shock protein 27 mRNA induction by osmotic stress and heat were similar with peak induction at 6 h. The mitogen activated protein kinase family of proteins plays an important part in the coordination of gene responses to various stress conditions. We have demonstrated that the p38 mitogen activated protein kinase was strongly activated by 200 mM and 300 mM sorbitol. The specific p38 mitogen activated protein kinase inhibitor PD169316 almost completely blocked heat shock protein 70 mRNA induction by 200 mM and 300 mM sorbitol and completely suppressed heat shock protein 27 mRNA induction with 200 mM sorbitol. PD169316 also counteracted upregulation of heat shock protein 70 levels by sorbitol. These data indicate that keratinocytes respond to osmotic stress by p38 mitogen activated protein kinase regulated induction of heat shock proteins. This molecular pathway may be relevant for the mechanisms regulating the response of human skin to variations in environmental humidity. Human skin is exposed to an environment that varies in humidity from 100 to 0%, leading to seasonal variations in the condition of the skin. Exposure to a low humidity environment creates an osmotic gradient across the stratum corneum, which is known to modulate cutaneous barrier function. Heat shock proteins protect against stress-induced destabilization of proteins. We investigated whether osmotic shock (sorbitol) induced a heat shock protein response in normal human keratinocytes, and used heat shock as a positive control. Both heat shock and osmotic stress (200 and 300 mM sorbitol) clearly induced heat shock proteins 70 and 27 mRNA levels. The induction of heat shock protein 70 mRNA levels by osmotic stress peaked at 16 h and persisted until 24 h, whereas upregulation of heat shock protein 70 mRNA levels by heat peaked at 2 h and returned to baseline levels by 6 h. Sorbitol also increased heat shock protein 70 levels in a concentration-dependent manner. The kinetics of heat shock protein 27 mRNA induction by osmotic stress and heat were similar with peak induction at 6 h. The mitogen activated protein kinase family of proteins plays an important part in the coordination of gene responses to various stress conditions. We have demonstrated that the p38 mitogen activated protein kinase was strongly activated by 200 mM and 300 mM sorbitol. The specific p38 mitogen activated protein kinase inhibitor PD169316 almost completely blocked heat shock protein 70 mRNA induction by 200 mM and 300 mM sorbitol and completely suppressed heat shock protein 27 mRNA induction with 200 mM sorbitol. PD169316 also counteracted upregulation of heat shock protein 70 levels by sorbitol. These data indicate that keratinocytes respond to osmotic stress by p38 mitogen activated protein kinase regulated induction of heat shock proteins. This molecular pathway may be relevant for the mechanisms regulating the response of human skin to variations in environmental humidity. enzyme immunoassay extracellular signal regulated kinase, GAPDH, glyceraldehyde phosphate dehydrogenase heat shock proteins heat shock transcription factor 1 c-Jun N-terminal kinase mitogen activated protein kinase Human epidermis is a multilayered epithelium that forms the interface between the environment and the organism, to protect the body both against excessive water loss as well as against penetration of potentially damaging substances. Environmental conditions, particularly changes in humidity, have an effect on skin condition. The terrestrial environment can vary in humidity from 0 to 100%. Exposure to a dry environment has profound effects on epidermal structure and function, which are even more pronounced when the barrier function of the stratum corneum is disrupted (by organic solvents or detergents) or impaired due to common dermatoses, including atopic eczema or psoriasis (Denda et al., 1998aDenda M. Sato J. Tsuchiya T. Elias P.M. Feingold K.R. Low humidity stimulates epidermal DNA synthesis and amplifies the hyperproliferative response to barrier disruption: implication for seasonal exacerbations of inflammatory dermatoses.J Invest Dermatol. 1998; 111: 873-878Crossref PubMed Scopus (225) Google Scholar). Exposure to a low humidity environment results in an enhancement of the epidermal barrier function, which can be considered as a homeostatic repair response, whereas barrier recovery is delayed in a high humidity environment (Denda et al., 1998bDenda M. Sato J. Masuda Y. et al.Exposure to a dry environment enhances epidermal permeability barrier function.J Invest Dermatol. 1998; 111: 858-863Crossref PubMed Scopus (201) Google Scholar). The epidermal release of pro-inflammatory cytokines such as interleukin-1 is amplified (Ashida et al., 2001Ashida Y. Ogo M. Denda M. Epidermal interleukin-1 generation is amplified at low humidity: implications for the pathogenesis of inflammatory dermatoses.Br J Dermatol. 2001; 144: 238-243Crossref PubMed Scopus (83) Google Scholar), and the contact hypersensitivity response is higher in a low humidity environment (Hosoi et al., 2000Hosoi J. Hariya T. Denda M. Tsuchiya T. Regulation of the cutaneous allergic reaction by humidity.Contact Dermatitis. 2000; 42: 81-84Crossref PubMed Scopus (42) Google Scholar). A hyperosmotic stimulus has been shown to produce a rapid transient increase in intracellular calcium in keratinocytes (Dascalu et al., 2000Dascalu A. Matithyou A. Oron Y. Korenstein R. A hyperosmotic stimulus elevates intracellular calcium and inhibits proliferation of a human keratinocyte cell line.J Invest Dermatol. 2000; 115: 714-718https://doi.org/10.1046/j.1523-1747.2000.00099.xCrossref PubMed Scopus (35) Google Scholar), whereas low humidity has been shown to stimulate epidermal DNA synthesis and modulate keratinocyte proliferation (Denda et al., 1998bDenda M. Sato J. Masuda Y. et al.Exposure to a dry environment enhances epidermal permeability barrier function.J Invest Dermatol. 1998; 111: 858-863Crossref PubMed Scopus (201) Google Scholar). Therefore, changes in environmental humidity may directly affect the behavior of the living cells in the epidermis. This study investigates the effect of the agent sorbitol, mimicking a drying osmotic environment, in cultured normal human keratinocytes, which represent the dividing and metabolically active cells of the epidermis. One of the consequences of osmotic shock is loss of cellular water and denaturation of intracellular proteins. Protein denaturation induces the synthesis of heat shock proteins (HSP). HSP are considered to be molecular chaperones that bind to unfolded and denatured proteins, promoting their (re-)folding and correct assembly (Beckmann et al., 1990Beckmann R.P. Mizzen L.A. Welch W.J. Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly.Science. 1990; 248: 850-854Crossref PubMed Scopus (1045) Google Scholar;De Maio, 1999De Maio A. Heat shock proteins: facts, thoughts, and dreams.Shock. 1999; 11: 1-12Crossref PubMed Scopus (504) Google Scholar;Feder and Hofmann, 1999Feder M.E. Hofmann G.E. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology.Annu Rev Physiol. 1999; 61: 243-282Crossref PubMed Scopus (3175) Google Scholar). The HSP response of keratinocytes to heat has been well characterized previously (Maytin et al., 1990Maytin E.V. Wimberly J.M. Anderson R.R. Thermotolerance and the heat shock response in normal human keratinocytes in culture.J Invest Dermatol. 1990; 95: 635-642Abstract Full Text PDF PubMed Scopus (52) Google Scholar;Maytin, 1992Maytin E.V. Differential effects of heat shock and UVB light upon stress protein expression in epidermal keratinocytes.J Biol Chem. 1992; 267: 23189-23196PubMed Google Scholar). It was our first aim to investigate whether osmotic shock induces the HSP response in keratinocytes. We studied the effect of osmotic stress on mRNA levels and protein levels of HSP. Two well-known HSP are HSP70 and HSP27. The 70 kDa family of HSP consists of constitutive and stress-inducible isoforms, which act as molecular chaperones (Tavaria et al., 1996Tavaria M. Gabriele T. Kola I. Anderson R.L. A hitchhiker's guide to the human Hsp70 family.Cell Stress Chaperones. 1996; 1: 23-28Crossref PubMed Scopus (325) Google Scholar). HSP27 encodes the 27 kDa HSP family or the small HSP, which function as molecular chaperones in thermotolerance (Jacob et al., 1993Jacob U. Gaestel M. Engel K. Buchner J. Small heat shock proteins are molecular chaperones.J Biol Chem. 1993; 268: 1517-1520PubMed Google Scholar;Fortin et al., 2000Fortin A. Raybaud-Diogene H. Tetu B. Huot J. Landry J. Deschenes R. Overexpression of the 27 kDa heat shock protein is associated with thermoresistance and chemoresistance but not with radioresistance.Int J Radiat Oncol Biol Phys. 2000; 46: 1259-1266https://doi.org/10.1016/S0360-3016(99)00410-1Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Furthermore, HSP27 expression is related to cell growth and differentiation (Jantschitsch et al., 1998Jantschitsch C. Kindas-Mügge I. Metze D. Amann G. Micksche M. Trautinger F. Expression of the small heat shock protein HSP 27 in developing human skin.Br J Dermatol. 1998; 139: 247-253Crossref PubMed Scopus (55) Google Scholar). The HSP response involves increased expression of genes encoding HSP. Increased gene expression is often regulated by the sequential activation of cytoplasmatic protein kinases. The mitogen activated protein kinase (MAPK) family of protein kinases plays an important part in coordinating gene responses to growth factors and various environmental stress conditions. Three structurally related but biochemically and functionally distinct MAPK signal transduction pathways have been identified and include p38 MAPK pathway, the c-jun N-terminal kinase (JNK) pathway and extracellular signal regulated (ERK) pathway (Su and Karin, 1996Su B. Karin M. Mitogen-activated protein kinase cascades and regulation of gene expression.Curr Opin Immunol. 1996; 8: 402-411Crossref PubMed Scopus (716) Google Scholar). p38 MAPK is activated by various environmental stresses, including cytokines and ultraviolet (UV). JNK is also activated by these environmental stress factors, but it participates in growth factor signaling as well. In general, ERK are primarily activated in response to growth factors and phorbol esters, whereas stress and inflammatory cytokines poorly activate the ERK. (Raingeaud et al., 1995Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine.J Biol Chem. 1995; 270: 7420-7426Crossref PubMed Scopus (2040) Google Scholar;Whitmarsh et al., 1995Whitmarsh A.J. Shore P. Sharrocks A.D. Davis R.J. Integration of MAP kinase signal transduction pathways at the serum response element.Science. 1995; 269: 403-407Crossref PubMed Scopus (880) Google Scholar;Canman and Kastan, 1996Canman C.E. Kastan M.B. Signal transduction. Three paths to stress relief.Nature. 1996; 384: 213-214Crossref PubMed Scopus (173) Google Scholar;Assefa et al., 1997Assefa Z. Garmyn M. Bouillon R. Merlevede W. Vandenheede J.R. Agostinis P. Differential stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in human keratinocytes.J Invest Dermatol. 1997; 108: 886-891Crossref PubMed Scopus (137) Google Scholar,Assefa et al., 2000Assefa Z. Vantieghem A. Garmyn M. et al.p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.J Biol Chem. 2000; 275: 21416-21421Crossref PubMed Scopus (141) Google Scholar;Peus et al., 1999Peus D. Remus A. Vasa R.A. Beyerle A. Meves A. Krautmacher C. Pittelkow M.R. UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.J Invest Dermatol. 1999; 112: 751-756Crossref PubMed Scopus (214) Google Scholar). Our second aim was to investigate the effect of osmotic stress on the MAPK pathway in keratinocytes and whether a specific MAPK signal transduction is involved in the upregulation of HSP after osmotic stress. Normal human keratinocytes were isolated from the foreskin of a young donor and grown in low-calcium (0.09 mM) keratinocyte serum-free medium (Gibco-BRL, Invitrogen, Merelbeke, Belgium) supplemented with bovine pituitary extract (50 µg per ml) and 5 ng human epidermal growth factor. Forty-eight hours before treatment cells were starved with epidermal growth factor-free keratinocyte serum-free medium, supplemented with 10 µg bovine pituitary extract (growth factor deprived medium). Keratinocytes were incubated with low calcium growth factor deprived medium (control), or with the same medium, made hypertonic by adding 50, 100, 200, or 300 mM Sorbitol (Sigma-Aldrich, Bornem, Belgium). Cells were harvested 2 h, 6 h, 16 h, and 24 h after addition of sorbitol, or after 4 h in the case of Hsp protein determination by enzyme immunoassay (EIA). As a positive control, cells were heat shocked at 45°C for 20 min and harvested at the same time-points as the sorbitol-treated cells. In a subset of experiments cells were, either temporarily treated with sorbitol for 2 h, or not starved prior to incubation with hyperosmotic medium. Results of these experiments are discussed, but not shown. The p38 MAPK inhibitor PD169316 was purchased from Calbiochem (Bierges, Belgium). Total RNA was isolated using Rneasy kit from Qiagen (Westburg, Heusden, The Netherlands). Twenty micrograms total RNA was fractionated by formaldehyde/agarose gel electrophoresis blotted to a nylon membrane and fixed by UV cross-linking, as described (Courtois et al., 1997Courtois S.J. Woodworth C.D. Degreef H. Garmyn M. Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.Exp Cell Res. 1997; 233: 135-144https://doi.org/10.1006/excr.1997.3537Crossref PubMed Scopus (27) Google Scholar). Prehybridization and hybridization were done in 50% formamide, 5 × Denhardt's, 6 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate, 50 mM sodium phosphate pH 7.0, denatured herring sperm DNA (200 µg per ml) at 42°C for 2–5 h. DNA probes were labeled by the random priming method with α-32P-deoxycytidine triphosphate. Blots were washed under increasingly stringent conditions (last step in 0.1 × sodium citrate/chloride buffer; 0.1% sodium dodecyl sulfate) and exposed for the appropriate duration. Autoradiography blots were compared by densitometry scanning. When the same blot was used for rehybridization it was stripped by two successive cycles of boiling in water containing 0.1% sodium dodecyl sulfate. The integrity of RNA samples was verified by ethidium bromide staining before blotting. The DNA probes that were used were: the human HSP70 cDNA and the human HSP27 cDNA probe (StressGen Biotechnologies Corp., Victoria, Canada). To normalize total RNA loading, blots were hybridized with the cDNA fragment of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1200 bp fragment cloned into PstI site of the vector pBR 322) (Yun et al., 1985Yun T.J. Sun X. Kao T. Reece K.S. Wu R. : Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.Nucleic Acids Res. 1985; 7: 2485-2488Google Scholar) Protein extracts were prepared as described (Assefa et al., 2000Assefa Z. Vantieghem A. Garmyn M. et al.p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.J Biol Chem. 2000; 275: 21416-21421Crossref PubMed Scopus (141) Google Scholar). The amount of HSP70 in cell lysates was assayed by EIA using a mouse monoclonal antibody precoated plate according to instructions from the supplier (StressGen Biotechnologies Corp.). Detection of the bound HSP70 is based on the subsequent recognition by a HSP70 specific, biotinylated rabbit polyclonal antibody. The biotinylated detector antibody is then bound by an avidin–horseradish peroxidase conjugate. Color development occurs after reaction with tetramethylbenzidine substrate, followed by the addition of acid stop solution to convert the end-point color to yellow and quantification by reading the absorbance at 450 nm for comparison with a standard curve. Protein extracts were prepared as described (Assefa et al., 2000Assefa Z. Vantieghem A. Garmyn M. et al.p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.J Biol Chem. 2000; 275: 21416-21421Crossref PubMed Scopus (141) Google Scholar). Protein concentration was determined using the BCA Protein Assay Reagent (Pierce Chemical Company, IL). Samples 100 µg protein from cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by wet electrotransfer on to nitrocellulose membrane (Hybond-C Super, Amersham, Rosendaal, the Netherlands). Blocking was performed in phosphate-buffered saline-0.1% Tween 20 supplemented with 5% nonfat dry milk for at least 1 h at 20°C. After four washings, the membrane was incubated overnight at 4°C in phosphate-buffered saline-3% bovine serum albumin with the primary antibody. Primary antibodies used were monoclonal antibodies, which specifically recognize the phosphorylated (activated) form of the MAPK, ERK, JNK, and p38: Anti-Active MAPK, for ERK1/2; Anti-Active JNK for JNK (Promega, Leiden, the Netherlands) and antiphospho-p38 antibody (New England Biolabs, Beverly, MA, USA) for p38 MAPK. After four washings in phosphate-buffered saline-0.1% Tween 20, the membrane was incubated for 1–2 h at 20°C with the peroxidase-conjugated secondary antibody and then autoradiographed using enhanced chemiluminescence as directed by the supplier (Amersham Life Sciences). Sorbitol, within the range of 200–300 mM, clearly upregulated HSP70 mRNA levels. HSP70 mRNA levels started to increase at 2 h. A clear increase in HSP70 mRNA levels was observed at 6 h, peak increase occurred at 16 h and increased mRNA levels were still present at the latest time-point studied (24 h). The positive control, heat shock, upregulated HSP70 mRNA levels earlier and more transiently (peaking at 2 h and almost disappeared by 6 h) Figure 1a, b. Both heat shock and sorbitol (100–300 mM) upregulated HSP27 mRNA levels, with peak mRNA levels at 6 h Figure 1a, c. The induction of HSP70 was confirmed at the protein level, using EIA Figure 2. Sorbitol-treated keratinocytes showed a concentration-dependent increase in HSP70 protein levels after 4 h of exposure. The HSP70 protein level obtained after exposure to 200 mM sorbitol was in the same range as the HSP70 level in the heat shocked control that was harvested at the same time. Keratinocytes, continuously exposed to 100, 200, and 300 mM sorbitol, were capable of complete recovery from osmotic stress, whereas exposure to 600 and 1000 mM sorbitol caused cell death. We evaluated the effect of a shorter sorbitol treatment (2 h) on HSP70 and HSP27 mRNA levels, to determine whether cells could recover from a shorter period of osmotic stress. Keratinocytes were treated with medium containing sorbitol (100, 200, 300, 600, and 1000 mM) for 2 h and then switched to old medium. In keratinocytes, exposed to 600 mM sorbitol for only a short period (2 h), we observed at 6 h a clear increase in HSP70 mRNA levels (more than 2-fold) (results not shown), which was not observed in keratinocytes that were continuously exposed to 600 mM sorbitol. The MAPK family of protein kinases plays an important part in coordinating gene responses to various environmental stress conditions, and osmotic stress is an important environmental stress for the keratinocyte. We investigated levels of activated p38 MAPK, JNK, and ERK at different time points, following treatment of keratinocytes with 200 and 300 mM sorbitol. p38 MAPK showed a strong and early (from 5 min onwards) activation, induced by 200 and 300 mM Sorbitol. p38 MAPK activation was more transient with 200 than with 300 mM sorbitol. At 6 h p38 MAPK activation after 200 mM sorbitol was almost down to zero. p38 MAPK activation by 300 mM sorbitol persisted until 16 h Figure 3. JNK was moderately and dose-dependently activated by 200 and 300 mM sorbitol from 30 min onwards. Under our experimental conditions, we could not demonstrate ERK1/2 activation by osmotic stress, although ERK1/2 was clearly activated by the addition of epidermal growth factor (100 ng per ml), a known strong inducer of ERK (results not shown). The p38 MAPK showed an early and strong activation by sorbitol concentrations that also induced increased HSP70 and HSP27 mRNA levels. Therefore, we further explored the involvement of p38 MAPK in HSP induction. We investigated whether inhibition of p38 MAPK activity with the specific inhibitor of p38 MAPK, PD169316 (Assefa et al., 2000Assefa Z. Vantieghem A. Garmyn M. et al.p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.J Biol Chem. 2000; 275: 21416-21421Crossref PubMed Scopus (141) Google Scholar), would affect a sorbitol-mediated increase in HSP70 and HSP27 mRNA levels. As shown in Figure 4, both upregulation of HSP70 and HSP27 mRNA levels by sorbitol was counteracted when cells were pretreated with PD169316. Fifteen micromol of PD169316 per liter downregulated HSP70 mRNA levels by 200 and 300 mM sorbitol with 70% and more than 90%, respectively, as measured densitometrically after correction for GAPDH. Fifteen micromol of PD169316 per liter completely suppressed the induction of HSP27 mRNA levels with 200 µM. The p38 MAPK inhibitor had no effect on HSP70 and HSP27 induction by heat (not shown). Counteraction of osmotic stress induced HSP70 was confirmed at the protein level, using EIA Figure 5. Sorbitol (100 mM) treated keratinocytes showed an increase in HSP70 protein levels after 4 h of exposure. Ten micromol of PD169316 per liter downregulated the induction of HSP70 protein levels by 100 mM sorbitol with 85%. Fifteen and 20 µM PD169316 completely suppressed the induction of HSP70 protein levels by 100 µM sorbitol. The p38 MAPK inhibitor had no effect on baseline protein levels of HSP70. This study is to our knowledge one of the first observations of an increased expression of HSP at mRNA and protein level caused by osmotic stress in human keratinocytes. The hyperosmotic agent sorbitol increases HSP70 mRNA levels with different kinetics than HSP27 mRNA levels. As a positive control, we used heat stress, a known inducer of HSP in mammalian cells, including keratinocytes. Upregulation of HSP70 mRNA levels by heat starts earlier and is more transient than upregulation of HSP70 mRNA levels by osmotic stress. Upregulation of HSP27 mRNA levels by heat and osmotic stress have similar kinetics. Previous studies have shown HSP induction by osmotic shock, predominantly in renal cells, which are exposed to large variations in osmotic stress caused by diuretic and antidiuretic fluctuations. (Cohen et al., 1991Cohen D.M. Wasserman J.C. Gullans S.R. Immediate early gene and HSP70 expression in hyperosmotic stress in MDCK cells.Am J Physiol. 1991; 261: C594-C601PubMed Google Scholar;Muller et al., 1996Muller E. Neuhofer W. Ohno A. Rucker S. Thurau K. Beck F.X. Heat shock proteins HSP25, HSP60, HSP72, HSP73 in isoosmotic cortex and hyperosmotic medulla of rat kidney.Pflugers Arch. 1996; 431: 608-617https://doi.org/10.1007/s004240050042Crossref PubMed Scopus (71) Google Scholar;Sheikh-Hamad et al., 1998Sheikh-Hamad D. Di Mari J. Suki W.N. Safirstein R. Watts B.A. Rouse D. p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells.J Biol Chem. 1998; 273: 1832-1837Crossref PubMed Scopus (163) Google Scholar). Likewise, a potential function for the HSP in the response of keratinocytes to osmotic stress can be envisioned from their capacity to maintain protein conformation (Beckmann et al., 1990Beckmann R.P. Mizzen L.A. Welch W.J. Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly.Science. 1990; 248: 850-854Crossref PubMed Scopus (1045) Google Scholar). It is therefore conceivable that in epidermal keratinocytes HSP70 and HSP27 could play a part in protein stabilization in the face of the elevated and potentially denaturing intracellular ionic concentrations that accompany acute volume changes elicited by a drying environment. The potential role of the HSP response elicited by heat in the cellular response of keratinocytes to UVB has been extensively studied. Conflicting results have been reported. On the one hand, heat pretreatment increases survival and decreases the number of sunburn cells, thus apoptotic keratinocytes, in UVB-irradiated keratinocytes. HSP70, but not HSP27, appears to be involved in this protective effect. (Maytin et al., 1993Maytin E.V. Murphy L.A. Merrill M.A. Hyperthermia induces resistance to ultraviolet light B in primary and immortalized epidermial keratinocytes.Cancer Res. 1993; 53: 4952-4959PubMed Google Scholar,Maytin et al., 1994Maytin E.V. Wimberly J.M. Kane K.S. Heat shock modulates UVB-induces cell death in human epidemal keratinocytes: evidence for a hyperthermia inducible protective response.J Invest Dermatol. 1994; 103: 547-553Abstract Full Text PDF PubMed Scopus (61) Google Scholar;Trautinger et al., 1995Trautinger F. Kindas-Mügge I. Barlan B. Neuner P. Knobler R.M. 72-kD heat shock protein is a mediator of resistance to ultraviolet light B.J Invest Dermatol. 1995; 105: 160-162Crossref PubMed Scopus (91) Google Scholar,Trautinger et al., 1996Trautinger F. Kindas-Mügge I. Knobler R.M. Hönigsmann H. Stress proteins in the cellular response to ultraviolet radiation.J Photochem Photobiol B Biol. 1996; 35: 141-148https://doi.org/10.1016/s1011-1344(96)07344-7Crossref PubMed Scopus (0) Google Scholar;Simon et al., 1995Simon M.M. Reikerstorfer A. Schwarz A. Krone C. Luger T.A. Jaattela M. Schwarz T. Heat shock protein 70 overexpression affects the response to ultraviolet light in murine fibroblasts. Evidence for increased cell viability and suppression of cytokine release.J Clin Invest. 1995; 95: 926-933Crossref PubMed Scopus (217) Google Scholar;Kindas-Mugge et al., 1996Kindas-Mugge I. Herbacek I. Jantschitsch C. Micksche M. Trautinger F. Modification of growth and tumorigenicity in epidermal cell lines by DNA-mediated gene transfer of heat shock protein 27 KD (hsp27).Cell Growth Differ. 1996; 7: 1167-1174PubMed Google Scholar). In contrast, a protective effect of heat pretreatment against UV damage has been questioned by others, because heat induction of the HSP response (HSP72), reduces nucleotide excision repair in UVB-irradiated keratinocytes and fibroblasts, possibly due to heat-induced damage of repair proteins (Schmidt-Rose et al., 1999Schmidt-Rose T. Pollet D. Will K. Bergemann J. Wittern K.-P. Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells.J Photochem Photobiol. 1999; 53: 144-152Crossref PubMed Scopus (26) Google Scholar). This reduction in nucleotide excision repair, together with a reduced apoptotic response, an escape mechanism for irreversibly damaged cells, could be potentially harmful and result in survival of cells that may give rise to cancer. Whether or not the osmotic stress response protects against UV damage remains to be investigated. In this study we clearly show that osmotic stress induces HSP mRNA and protein levels in human keratinocytes in a concentration-dependent manner. A more sustained upregulation of HSP70 mRNA levels after osmotic stress than after heat shock is of particular interest in view of a potential photoprotective effect. To explore this possible photoprotective effect further, investigations on keratinocyte survival, sunburn cell induction, but also on DNA damage and repair, is required. As increased gene expression is often regulated by the sequential activation of cytoplasmatic protein kinases, we investigated the effect of osmotic stress on activation of the MAPK family of protein kinases. ERK, JNK, and p38 MAPK are activated by osmotic stress in other cellular systems (Itoh et al., 1994Itoh T. Yamauchi A. Miyai A. Yokoyama K. Kamada T. Ueda N. Fujiwara Y. Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells.J Clin Invest. 1994; 93: 2387-2392Crossref PubMed Scopus (117) Google Scholar;Galcheva-Gargova et al., 1994Galcheva-Gargova Z. Dérijard B. Wu I.-H. Davis R.J. An osmosensing signal tranduction pathway in mammalian cells.Science. 1994; 265: 806-808Crossref PubMed Scopus (531) Google Scholar;Terada et al., 1994Terada Y. Tomita K. Homma M.K. et al.Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells.J Biol Chem. 1994; 269: 31296-31301PubMed Google Scholar;Matsuda et al., 1995Matsuda S. Kawasaki H. Moriguchi T. Gotoh Y. Nishida E. Activation of protein kinase cascades by osmotic shock.J Biol Chem. 1995; 270: 12781-12786Crossref PubMed Scopus (149) Google Scholar;Sheikh-Hamad et al., 1998Sheikh-Hamad D. Di Mari J. Suki W.N. Safirstein R. Watts B.A. Rouse D. p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells.J Biol Chem. 1998; 273: 1832-1837Crossref PubMed Scopus (163) Google Scholar). In our experimental conditions, sorbitol has no effect on ERK activation and only a moderate effect on JNK activation. In contrast, sorbitol preferentially activates p38 MAPK in human keratinocytes. To confirm further the involvement of p38 MAPK in osmotic-stress-induced increase in HSP at the mRNA and protein level in human keratinocytes, we investigated the effect of PD169316. This potent and specific inhibitor of the p38 MAPK is an imidazole-based compound used both in vivo and in vitro to confirm the specific involvement of the p38 MAPK pathway in a variety of cellular physiologic processes (Boehm et al., 1996Boehm J.C. Smietana J.M. Sorenson M.E. et al.1-substituted 4-aryl-5-pyridinylimidazoles: a new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency.J Med Chem. 1996; 39: 3929-3937Crossref PubMed Scopus (130) Google Scholar). Incubation of cells with this compound inhibited the Myelin basic protein (MBP)-phosphotransferase activity of p38 MAPK in immunoprecipitation assays without affecting the activities of JNK1 and ERK2 (Assefa et al., 2000Assefa Z. Vantieghem A. Garmyn M. et al.p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.J Biol Chem. 2000; 275: 21416-21421Crossref PubMed Scopus (141) Google Scholar). We showed that PD169316 counteracts the upregulation of HSP70 mRNA levels and HSP27 mRNA levels by osmotic stress in normal human keratinocytes. PD169316 also counteracts upregulation of Hsp70 protein levels by osmotic stress in normal human keratinocytes. These findings suggest that p38 MAPK activity is essential for osmotically induced upregulation of HSP, which may contribute to the adaptation of keratinocytes to osmotic stress. The p38 inhibitor PD169316 did not suppress heat shock induced HSP70 and HSP27 mRNA levels (results not shown) suggesting that the signal transduction pathways mediating increased HSP70 and HSP27 mRNA levels by different stresses are divergent: the pathways mediating osmotic-stress induced increase in HSP70 and HSP27 mRNA levels are p38 MAPK dependent, whereas those mediating thermal induced upregulation of HSP70 and HSP27 mRNA levels are not. In other cell types, p38 MAPK is involved in HSP27 activation by phosphorylation of the HSP27 protein, thereby mediating its homeostatic function at the actin-cytoskeleton level. (Guay et al., 1997Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.J Cell Sci. 1997; 110: 357-368PubMed Google Scholar;Schafer et al., 1998Schafer C. Ross S.E. Bragado M.J. Groblewski G.E. Ernst S.A. Williams J.A. A role for the p38 mitogen-activated protein kinase/Hsp27 pathway in cholecystokinin-induced changes in the actin cytoskeleton in rat pancreatic acini.J Biol Chem. 1998; 273: 24173-24180Crossref PubMed Scopus (141) Google Scholar). The importance of this actin signaling pathway depends on HSP27 expression levels, which in human keratinocytes are also regulated by p38 MAPK. It remains to be determined whether in primary keratinocytes osmotic stress induces HSP27 phosphorylation and whether this process is p38 MAPK dependent, as very recently shown for UVB (Wong et al., 2000Wong J.W. Shi B. Farboud B. McClaren M. Shibamoto T. Cross C.E. Isseroff R.R. Ultraviolet B-mediated phosphorylation of the small heat shock protein HSP27 in human keratinocytes.J Invest Dermatol. 2000; 115: 427-434Crossref PubMed Scopus (42) Google Scholar). If confirmed, these findings suggest that in human skin p38 MAPK would play a key part in safeguarding the actin skeleton upon osmotic stress by both regulating expression and activation of HSP27. We cannot completely exclude an additional involvement of the JNK pathway in osmotic-stress-induced upregulation of HSP70 and HSP27 mRNA levels in human keratinocytes. Similar to what we observed in our keratinocytes, also in other cellular systems JNK was shown to be activated by osmotic shock (Galcheva-Gargova et al., 1994Galcheva-Gargova Z. Dérijard B. Wu I.-H. Davis R.J. An osmosensing signal tranduction pathway in mammalian cells.Science. 1994; 265: 806-808Crossref PubMed Scopus (531) Google Scholar;Matsuda et al., 1995Matsuda S. Kawasaki H. Moriguchi T. Gotoh Y. Nishida E. Activation of protein kinase cascades by osmotic shock.J Biol Chem. 1995; 270: 12781-12786Crossref PubMed Scopus (149) Google Scholar). In our experimental conditions, however, p38 MAPK was preferentially activated by osmotic stress and the highly specific p38 inhibitor almost completely abolished upregulation of HSP70 and HSP27 mRNA levels by sorbitol. The p38 MAPK pathway is therefore the major pathway of osmotic-stress-induced upregulation of HSP70 and HSP27 mRNA levels in primary keratinocytes. Initially we starved the keratinocytes for 48 h in order to exclude interference of growth factors, which are known stimulators of the ERK pathway and to a lesser extent the JNK pathway. In a subset of experiments keratinocytes were not starved prior to osmotic shock (results not shown), yet they display similar kinetics of sorbitol-induced upregulation of HSP70 and HSP27 mRNA levels as in the starved cells. Thus, growth factors did not significantly interfere with the osmotic stress-induced increase in HSP mRNA levels in human keratinocytes. This finding is in accordance with the lack of ERK activation by osmotic stress in human keratinocytes and further suggests that ERK is not a component of the osmotic stress-induced signal transduction pathway in human keratinocytes. In conclusion, we have shown that osmotic shock induces increased expression of HSP at the mRNA and protein level in human keratinocytes. For HSP70, the upregulation of its message level occurs later during osmotic treatment and is more persistent than the increase of its mRNA level by heat. The pattern of osmotic stress-induced and heat-induced increase in HSP27 mRNA levels are quite similar. The p38 MAPK is preferentially activated by osmotic stress and the p38 MAPK inhibitor PD169316 counteracts osmotic shock-induced HSP expression both at the mRNA and protein level, strongly indicating that p38 MAPK is involved in osmotically induced upregulation of HSP, which may contribute to the adaptation of keratinocytes to osmotic stress. Osmotically induced expression of HSP70 and HSP27 and their upstream pathways deserve further investigation as a model to study the effects of a drying environment on skin cells. This work was supported by (Estée Lauder Companies) and an OT/96/27 grant of the University of Leuven, Belgium. M.G. is clinical research associate from the FWO. We thank Mary Matsui for critically reading the manuscript and Inge Daris for her excellent technical assistance.