The Sex Steroid Precursor DHEA Accelerates Cutaneous Wound Healing Via the Estrogen Receptors
2005; Elsevier BV; Volume: 125; Issue: 5 Linguagem: Inglês
10.1111/j.0022-202x.2005.23926.x
ISSN1523-1747
AutoresStuart J. Mills, Jason J. Ashworth, Stephen C. Gilliver, Matthew J. Hardman, Gillian S. Ashcroft,
Tópico(s)Hormonal and reproductive studies
ResumoAge-related impaired wound healing states lead to substantial morbidity and cost, with treatment in the USA resulting in an expenditure of over $9 billion per annum. Dehydroepiandrosterone (DHEA) is a ubiquitous adrenal hormone with immunomodulatory properties whose levels decline significantly with advanced age in humans. Conversion of DHEA locally to downstream steroid hormones leads to estrogenic and/or androgenic effects which may be important in age-related skin homeostasis, and which would avoid systemic adverse effects related to estrogen. We report that systemic DHEA levels are strongly associated with protection against chronic venous ulceration in humans. DHEA accelerated impaired healing in an impaired healing model (mice rendered hypogonadal) associated with increased matrix deposition and dampens the exaggerated inflammatory response. Such effects were mediated by local conversion of DHEA to estrogen, acting through the estrogen receptor, and vitro studies suggest a direct effect on specific pro-inflammatory cytokine production by macrophages via mitogen activated kinase (MAP) and phosphatidylinositol 3 (PI3) kinase pathways. In addition, we show that local injection of DHEA accelerates impaired healing in an ageing mouse colony. We suggest that exogenous application of DHEA accelerates impaired wound repair, results which may be applicable to the prophylaxis and treatment of human impaired wound healing states. Age-related impaired wound healing states lead to substantial morbidity and cost, with treatment in the USA resulting in an expenditure of over $9 billion per annum. Dehydroepiandrosterone (DHEA) is a ubiquitous adrenal hormone with immunomodulatory properties whose levels decline significantly with advanced age in humans. Conversion of DHEA locally to downstream steroid hormones leads to estrogenic and/or androgenic effects which may be important in age-related skin homeostasis, and which would avoid systemic adverse effects related to estrogen. We report that systemic DHEA levels are strongly associated with protection against chronic venous ulceration in humans. DHEA accelerated impaired healing in an impaired healing model (mice rendered hypogonadal) associated with increased matrix deposition and dampens the exaggerated inflammatory response. Such effects were mediated by local conversion of DHEA to estrogen, acting through the estrogen receptor, and vitro studies suggest a direct effect on specific pro-inflammatory cytokine production by macrophages via mitogen activated kinase (MAP) and phosphatidylinositol 3 (PI3) kinase pathways. In addition, we show that local injection of DHEA accelerates impaired healing in an ageing mouse colony. We suggest that exogenous application of DHEA accelerates impaired wound repair, results which may be applicable to the prophylaxis and treatment of human impaired wound healing states. androgen receptor dehydroepiandrosterone estrogen receptor interleukin 6 mitogen activated kinase macrophage migration inhibitory factor ovariectomized phosphatidylinositol 3 protein kinase C tumor necrosis factor-alpha Circulating levels of dehydroepiandrosterone (DHEA) and its sulfate ester, DHEA sulfate (DHEAS), decline progressively and markedly with age, in distinct contrast to other adrenal steroids such as glucocorticoids, whose serum levels remain relatively well-preserved with age (Labrie et al., 1998Labrie F. Belanger A. Luu-The V. et al.DHEA and the intracrine formation of androgens and estrogens in peripheral target tissues: Its role during aging.Steroids. 1998; 63: 322-328Crossref PubMed Scopus (276) Google Scholar). Human epidemiologic studies suggest that an elevated serum DHEAS level may confer beneficial effects in terms of increased longevity and prevention of heart disease (Jarrar et al., 2000Jarrar D. Wang P. Cioffi W.G. et al.Mechanisms of the salutary effects of dehydroepiandrosterone after trauma-haemorrhage: Direct or indirect effects on cardiac and hepatocellular functions?.Arch Surg. 2000; 135 (discussion 422–413): 416-422Crossref PubMed Scopus (41) Google Scholar). Thus serum DHEA and/or DHEAS may represent biomarkers of healthy ageing. Numerous animal studies have convincingly demonstrated the beneficial effects of DHEA administration in preventing obesity, diabetes, cancer, and heart disease, in enhancing the immune system, and even in prolonging the life-span (Angele et al., 1998Angele M.K. Catania R.A. Ayala A. et al.Dehydroepiandrosterone: An inexpensive steroid hormone that decreases the mortality due to sepsis following trauma-induced haemorrhage.Arch Surg. 1998; 133: 1281-1288Crossref PubMed Scopus (89) Google Scholar). Collectively, these observations have led investigators to speculate that some of the degenerative changes associated with human ageing may be related to a progressive deficit in circulating levels of DHEA and/or DHEAS. A number of human studies have recently reported the beneficial effects of DHEA on the human immune system in restoring a competent immune response, and reducing atherosclerosis, possibly by inhibiting platelet aggregation and altering the circulating levels of proteases (Jesse et al., 1995Jesse R.L. Loesser K. Eich D.M. et al.Dehydroepiandrosterone inhibits human platelet aggregation in vitro and in vivo.Annu N Y Acad Sci. 1995; 774: 281-290Crossref PubMed Scopus (68) Google Scholar; Angele et al., 1998Angele M.K. Catania R.A. Ayala A. et al.Dehydroepiandrosterone: An inexpensive steroid hormone that decreases the mortality due to sepsis following trauma-induced haemorrhage.Arch Surg. 1998; 133: 1281-1288Crossref PubMed Scopus (89) Google Scholar). How DHEA exerts its effects are, as yet, unknown, but may act by influencing several key metabolic enzymes such as glucose-6-phosphate dehydrogenase, and by its conversion to androgens and estrogens (Schwartz and Pashko, 2004Schwartz A.G. Pashko L.L. Dehydroepiandrosterone, glucose-6-phosphate dehydrogenase, and longevity.Ageing Res Rev. 2004; 3: 171-187Crossref PubMed Scopus (93) Google Scholar). Some actions of DHEA may not involve a classic steroid receptor or gene activation, as evidenced by the fact that DHEA can inhibit platelet aggregation (Jesse et al., 1995Jesse R.L. Loesser K. Eich D.M. et al.Dehydroepiandrosterone inhibits human platelet aggregation in vitro and in vivo.Annu N Y Acad Sci. 1995; 774: 281-290Crossref PubMed Scopus (68) Google Scholar). It could be postulated that DHEA, as a precursor of androgens and estrogens, may influence wound healing in the elderly by stimulating the rate of wound repair. This view is supported by recent studies implicating topical DHEA as a mediator of tissue repair in UV-damaged intact skin (Shin et al., 2005Shin M.H. Rhie G.E. Park C.H. et al.Modulation of collagen metabolism by the topical application of dehydroepiandrosterone to human skin.J Invest Dermatol. 2005; 124: 315-323Crossref PubMed Scopus (59) Google Scholar). Impaired wound healing states, both acute wounds which fail to heal and chronic ulcers, are characterized by excessive leukocytosis and subsequently enhanced proteolytic degradation of matrix constituents (Ashcroft et al., 1997aAshcroft G.S. Dodsworth J. van Boxtel E. et al.Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels.Nat Med. 1997; 3: 1209-1215Crossref PubMed Scopus (439) Google Scholar,Ashcroft et al., 1999bAshcroft G.S. Yang X. Glick A.B. et al.Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.Nat Cell Biol. 1999; 1: 260-266Crossref PubMed Scopus (740) Google Scholar ,2000; Herrick et al., 1997Herrick S. Ashcroft G. Ireland G. et al.Up-regulation of elastase in acute wounds of healthy aged humans and chronic venous leg ulcers are associated with matrix degradation.Lab Invest. 1997; 77: 281-288PubMed Google Scholar). Accumulating evidence implicates inflammation as a causative factor in delayed healing, and suggests that, in the absence of infection, the inflammatory response is inappropriately excessive. A large number of clinical and experimental studies have shown that low systemic levels of DHEA are associated with a variety of inflammatory disorders including SLE, systemic sclerosis and arthritis (Chen and Parker, 2004Chen C.C. Parker Jr, C.R. Adrenal androgens and the immune system.Semin Reproductive Med. 2004; 22: 369-377Crossref PubMed Scopus (84) Google Scholar). Despite rodents producing no endogenous source of DHEA, they possess the necessary enzymatic machinery to convert exogenous DHEA to sex steroids. DHEA supplementation has been shown to be beneficial in murine models of inflammation and aging, including reducing inflammation and tissue destruction resulting from cutaneous thermal injury (Araneo et al., 1995Araneo B.A. Ryu S.Y. Barton S. et al.Dehydroepiandrosterone reduces progressive dermal ischemia caused by thermal injury.J Surg Res. 1995; 59: 250-262Abstract Full Text PDF PubMed Scopus (25) Google Scholar). One common downstream effect of DHEA in these systems appears to be inhibition of NF-κB DNA binding activity, resulting in dampened gene transcription of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) (Padgett and Loria, 1998Padgett D.A. Loria R.M. Endocrine regulation of murine macrophage function: effects of dehydroepiandrosterone, androstenediol, and androstenetriol.J Neuroimmunol. 1998; 84: 61-68Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Our previous studies have documented a positive role for topical and systemic estrogen on age-impaired wound healing (Ashcroft et al., 1997aAshcroft G.S. Dodsworth J. van Boxtel E. et al.Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels.Nat Med. 1997; 3: 1209-1215Crossref PubMed Scopus (439) Google Scholar,Ashcroft et al., 1999aAshcroft G.S. Greenwell-Wild T. Horan M.A. et al.Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response.Am J Pathol. 1999; 155: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Derivatives of DHEA such as estrogens, androstenedione, and 17-β androstenediol play an important immunoregulatory role, and recently DHEA has been shown to mediate its effects on macrophages via aromatase-induced conversion to sex steroids including estrogen (Schmidt et al., 2000Schmidt M. Kreutz M. Loffler G. et al.Conversion of dehydroepiandrosterone to downstream steroid hormones in macrophages.J Endocrinol. 2000; 164: 161-169Crossref PubMed Scopus (117) Google Scholar). DHEA is a potentially interesting candidate involved in the modulation of tissue repair, since its actions appear to circumvent a number of the systemic adverse effects attributable to estrogen such as tumorigenesis (Labrie et al., 2003Labrie F. Luu-The V. Labrie C. et al.Endocrine and intracrine sources of androgens in women: inhibition of breast cancer and other roles of androgens and their precursor dehydroepiandrosterone.Endocrine Rev. 2003; 24: 152-182Crossref PubMed Scopus (430) Google Scholar). This study aims to determine the pharmacological effects of DHEA on age-impaired wound healing and investigates the specific downstream mediators of DHEA activity on in vivo and in vitro functions. In order to determine a role for DHEA in impaired human healing responses, we monitored systemic DHEA levels in control aged human subjects and those with healed or pre-existing chronic venous ulcers. By ELISA, serum DHEA levels were significantly decreased in both male and female aged human subjects with, or who previously had suffered from, chronic venous ulceration compared to healthy controls (Figure 1a). These novel data suggested a potential association between impaired human wound healing and DHEA levels, and prompted an assessment of a causal role for DHEA in an experimental model of aberrant healing. Following full-thickness dorsal incisional wounding of mice deficient in estrogen and wild-type intact littermates, a markedly impaired rate of wound healing was readily apparent in the estrogen-deficient animals at day 3 post-wounding, as previously reported (Ashcroft et al., 1997aAshcroft G.S. Dodsworth J. van Boxtel E. et al.Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels.Nat Med. 1997; 3: 1209-1215Crossref PubMed Scopus (439) Google Scholar) (Figure 1b). Intact and ovariectomized (OVX) mice had no detectable levels of systemic DHEA consistent with the absence of adrenally derived DHEA production in rodents (data not shown). Systemic treatment with DHEA at the time of wounding (day 0) showed a non-significant decrease in wound areas, however in a concentration-dependent fashion, three consecutive treatments (d0, d1, d2) resulted in a highly significant acceleration of healing and reduction in wound area (Figure 1b). Not only were the wounds enlarged in the estrogen-deficient mice, but there was a substantial increase in cells per unit area reflecting a local accumulation of leukocytes as we have observed previously (Ashcroft et al., 1997aAshcroft G.S. Dodsworth J. van Boxtel E. et al.Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels.Nat Med. 1997; 3: 1209-1215Crossref PubMed Scopus (439) Google Scholar; Ashcroft and Mills, 2002Ashcroft G.S. Mills S.J. Androgen receptor-mediated inhibition of cutaneous wound healing.J Clin Invest. 2002; 110: 615-624Crossref PubMed Scopus (224) Google Scholar). Immunolocalization using specific neutrophil and macrophage markers indicated that both cell types were significantly reduced in the wounds of the OVX DHEA-treated mice compared to the OVX vehicle-treated mice (Figure 1c). Both a modest reduction in cell numbers, and DHEA effects on macrophage actions, may contribute to accelerated repair. In addition, collagen I levels, as determined by immunohistochemistry, were increased following DHEA treatment (Figure 1c). In order to further delineate the actions of DHEA on tissue responses, we investigated if conversion of DHEA to downstream metabolites was necessary for its effects. Following co-administration of an aromatase inhibitor, arimidex, in conjunction with DHEA treatment, we observed that DHEA no longer reversed the inhibitory effects in healing attributed to estrogen-depletion (Figure 2a). Arimidex alone had no effect on any parameter measured (data not shown). The dampening of the local inflammatory response by DHEA was also reversed by arimidex, both for macrophages and neutrophils (Figure 2b, c). These data implicated the conversion of DHEA by aromatase to estriol and subsequently estradiol as an essential prerequisite for DHEA's impact upon in vivo healing and inflammation. We reasoned that, since estrogenic conversion was an important step, specific pro-inflammatory genes regulated by estrogen would also be influenced by DHEA in vivo. DHEA inhibited macrophage migration inhibitory factor (MIF), IL-6 and TNF-α both at the protein and mRNA levels (Figure 3a–c), and this effect was globally reversed by arimidex treatment. Flutamide, an androgen receptor (AR) antagonist, had no effect on DHEA's actions (data not shown).Figure 3Administration of systemic dehydroepiandrosterone (DHEA) reduced wound tissue levels of pro-inflammatory cytokines. Cell counts (graphs) for (a) macrophage migration inhibitory factor (MIF), (b) tumor necrosis factor-alpha (TNF-α) and (c) interleukin 6 (IL-6) were significantly reduced in day 3 wounds where mice had been treated with DHEA (three doses), and arimidex reversed the effects of DHEA in all cases. Results shown are mean±SEM. *p<0.05, **p<0.01, n=6 per group. Panels show representative immunohistochemical staining of cells expressing (a) MIF, (b) TNF-α and (c) IL-6, illustrating reduced numbers of positively-staining cells (arrows) following DHEA treatment, and was confirmed by western blotting of wound tissue (β-actin acted as loading control). Graphs (on right of panel) represent real time PCR for MIF, TNF-α and IL-6 (all repeated in triplicate). Results shown are mean±SEM. *p<0.05. n=6 per group. Ovariectomized (OVX), OVX vehicle treated.View Large Image Figure ViewerDownload (PPT) Classically, estrogen acts by binding specific intracellular receptors, ER-α and ER-β, which then act as transcription factors, or influence other signalling pathways such as MAP kinase. Recent studies have highlighted the tissue-specific expression of such receptors and the regulation of immune function in vivo dependent upon the presence of the estrogen receptor (Jarrar et al., 2000Jarrar D. Wang P. Cioffi W.G. et al.Mechanisms of the salutary effects of dehydroepiandrosterone after trauma-haemorrhage: Direct or indirect effects on cardiac and hepatocellular functions?.Arch Surg. 2000; 135 (discussion 422–413): 416-422Crossref PubMed Scopus (41) Google Scholar). Estrogen, however, may also act through non-classical membrane receptors leading to rapid intracellular responses (Simoncini et al., 2003Simoncini T. Mannella P. Fornari L. et al.Dehydroepiandrosterone modulates endothelial nitric oxide synthesis via direct genomic and nongenomic mechanisms.Endocrinology. 2003; 144: 3449-3455Crossref PubMed Scopus (159) Google Scholar). We further investigated the mechanisms underlying the responses to DHEA by utilizing a systemic antagonist to both ER-α and ER-β. ICI 182,780 blocked the effects of DHEA on wound repair (Figure 4a), and significantly reversed the effects of DHEA on macrophage numbers (Figure 4b), and neutrophil cell counts (data not shown). Since the inflammatory response was dampened significantly in the DHEA-treated animals, we investigated whether DHEA had direct effects on macrophage responses, specifically on pro-inflammatory gene expression. In vitro studies confirmed that DHEA inhibited macrophage protein and mRNA production of IL-6, MIF and TNF-α (Figure 4c, d), and that DHEA's effects were dependent upon conversion to estrogen since arimidex reversed DHEA's actions (Figure 4c, d). ICI 182 treatment reversed the effects of DHEA on MIF expression, but not IL-6 and TNF-α (Figure 4c, d), suggesting that the latter effects were mediated by non-classical pathways. Since MAP/PI3 kinase pathways are important in immune/inflammatory responses we determined the levels of ERK1/2 and pERK in wound tissue and showed that estrogen depletion markedly increased protein levels of this intermediate, and DHEA reversed this effect (Figure 5a). Inhibitors of both MAP kinase and PI3 kinase pathways reversed the effects of DHEA on macrophage MIF levels, but not TNF-α (Figure 5b, c) and IL-6 (data not shown), suggesting involvement of these pathways in specific cellular responses to DHEA relating to ER activation. The reduction of DHEA with increasing age in humans has been associated with a number of pathological conditions, including skin ageing. Since our data suggested that DHEA levels were reduced in human subjects predisposed to impaired healing conditions, and DHEA could directly regulate wound repair in an impaired healing murine model of estrogen depletion, we reasoned that DHEA may have a role in reversing the delayed healing we have previously observed in an aging mouse colony (Ashcroft et al., 1997bAshcroft G.S. Horan M.A. Ferguson M.W. Aging is associated with reduced deposition of specific extracellular matrix components, an up-regulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model.J Invest Dermatol. 1997; 108: 430-437Crossref PubMed Scopus (180) Google Scholar). Young (6 weeks) and aged (30 month) mice were treated with DHEA injection into the skin immediately prior to wounding. Wound areas were increased in the aged mice compared to the young at early time-points (Figure 6a, b). DHEA had no effect on wound healing in the young mice from day 3 to 14 post-wounding however DHEA markedly reduced wound areas in the aged mice at days 3 and 7 post-wounding (Figure 6a, b). Collagen deposition was increased following DHEA administration in the aged mice at day 7 post-wounding (Figure 6c). DHEA and its sulphate ester are the most abundant circulating sex hormones in humans (Baulieu et al., 1965Baulieu E.E. Corpechot C. Dray F. et al.An adrenal-secreted "androgen" dehydroepiandrostereone sulphate. Its metabolism and a tentative generalization on the metabolism of the other steroid conjugates in man.Recent Prog Hormone Res. 1965; 21: 411-500PubMed Google Scholar). The new area of endocrinology termed "intracrinology" has made great progress following the cloning and characterization of most of the enzymes responsible for transforming DHEA into androgens and estrogens in peripheral target tissues, where the locally produced sex steroids are exerting their action often within their cell of origin. In a series of animal models, DHEA has been shown to inhibit breast cancer, to influence the immune response, and to stimulate positive estrogenic actions such as increased bone mineral density without predisposing to endometrial cancer. Thus the advantage of DHEA compared to estrogen and androgens is that DHEA is only converted to its active metabolites in the specific target tissues where the appropriate enzymatic machinery exists, thus limiting the adverse effects of systemic hormone treatment. DHEA levels from 60–70 y decline by 70% compared to 20–30 y, with relatively smaller changes occurring after 60 y (Labrie et al., 1997Labrie F. Belanger A. Cusan L. et al.Marked decline in serum concentrations of adrenal C19 sex steroid precursors and conjugated androgen metabolites during aging.J Clin Endocrinol Metab. 1997; 82: 2396-2402Crossref PubMed Scopus (528) Google Scholar). We report that highly significant systemic differences in DHEA levels in the elderly are associated with the predisposition to impaired chronic wound healing states. These data suggest that, in addition to age, genetic and physiological factors can influence DHEA levels, and implicate DHEA as a potential modulator of skin physiology and repair in humans. Despite DHEA's wide-ranging effects in age-related processes, and its putative protective role following brain injury (Hoffman et al., 2003Hoffman S.W. Virmani S. Simkins R.M. Stein D.G. The delayed administration of dehydroepiandrosterone sulfate improves recovery of function after traumatic brain injury in rats.J Neurotrauma. 2003; 20: 859-870Crossref PubMed Scopus (35) Google Scholar) only one previous study has examined its potential role in cutaneous wound healing, relating to thermal injury.Araneo et al., 1995Araneo B.A. Ryu S.Y. Barton S. et al.Dehydroepiandrosterone reduces progressive dermal ischemia caused by thermal injury.J Surg Res. 1995; 59: 250-262Abstract Full Text PDF PubMed Scopus (25) Google Scholar reported that DHEA influenced the progressive dermal ischaemia occurring following thermal injury to the skin, possible via a non-ER/AR mechanism. We show that DHEA can accelerate wound repair in an impaired model of healing secondary to estrogen-depletion, which mimics age-related healing (Ashcroft et al., 1997aAshcroft G.S. Dodsworth J. van Boxtel E. et al.Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels.Nat Med. 1997; 3: 1209-1215Crossref PubMed Scopus (439) Google Scholar, Ashcroft et al., 1997bAshcroft G.S. Horan M.A. Ferguson M.W. Aging is associated with reduced deposition of specific extracellular matrix components, an up-regulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model.J Invest Dermatol. 1997; 108: 430-437Crossref PubMed Scopus (180) Google Scholar). DHEA dampens the local inflammatory response and reduces the tissue levels of a number of pro-inflammatory cytokines. Our previous studies have shown that the excessive inflammatory response occurring in OVX mice and in the elderly leads to enhanced proteolysis (Ashcroft et al., 1997cAshcroft G.S. Horan M.A. Herrick S.E. et al.Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans.Cell Tissue Research. 1997; 290: 581-591Crossref PubMed Scopus (149) Google Scholar). In this regard it is interesting to note thatShin et al., 2005Shin M.H. Rhie G.E. Park C.H. et al.Modulation of collagen metabolism by the topical application of dehydroepiandrosterone to human skin.J Invest Dermatol. 2005; 124: 315-323Crossref PubMed Scopus (59) Google Scholar have recently reported that DHEA can reduce MMP-1 expression in aged skin, and that we show DHEA-mediated increased matrix deposition. DHEA may modulate wound healing via its peripheral conversion to estrogens or androgens, since it is known that both can influence repair and inflammation with estrogen stimulating healing and androgens inhibiting repair. In addition it has been suggested that DHEA may act upon, as yet unidentified, specific receptors inducing both genomic and non-genomic effects (Meikle et al., 1992Meikle A.W. Dorchuck R.W. Araneo B.A. et al.The presence of a dehydroepiandrosterone-specific receptor binding complex in murine T cells.J Steroid Biochem Mol Biol. 1992; 42: 293-304Crossref PubMed Scopus (180) Google Scholar; Ripp et al., 2003Ripp S.L. Falkner K.C. Pendleton M.L. et al.Regulation of CYP2C11 by dehydroepiandrosterone and peroxisome proliferators: Identification of the negative regulatory region of the gene.Mol Pharmacol. 2003; 64: 113-122Crossref PubMed Scopus (28) Google Scholar; Simoncini et al., 2003Simoncini T. Mannella P. Fornari L. et al.Dehydroepiandrosterone modulates endothelial nitric oxide synthesis via direct genomic and nongenomic mechanisms.Endocrinology. 2003; 144: 3449-3455Crossref PubMed Scopus (159) Google Scholar). We utilized an impaired wound-healing model involving estrogen-depletion in order to dissect apart the roles of active metabolites and specific receptors. Our data suggest that DHEA influences repair and local inflammation via its conversion to estrogen and subsequently through the ER. Many factors contribute to immunosenescence resulting in increased mortality and morbidity in the elderly (Emmerling et al., 1979Emmerling P. Hof H. Finger H. Age-related defense against infection with intracellular pathogens.Gerontology. 1979; 25: 327-336Crossref PubMed Scopus (28) Google Scholar). Age-related alterations in the functional capacity of macrophages are likely to contribute significantly to loss of natural immunity with dysregulated cytokine production and antigen-presenting ability (Vetvicka et al., 1985Vetvicka V. Tlaskalova-Hogenova H. Pospisil M. Impaired antigen presenting function of macrophages from aged mice.Immunol Invest. 1985; 14: 105-114Crossref PubMed Scopus (30) Google Scholar; Bradley et al., 1989Bradley S.F. Vibhagool A. Kunkel S.L. et al.Monokine secretion in aging and protein malnutrition.J Leukocyte Biol. 1989; 45: 510-514PubMed Google Scholar). Specifically, a defective PKC system underlies the age-associated impairment of a number of these processes (Corsini et al., 1999Corsini E. Battaini F. Lucchi L. et al.A defective protein kinase C anchoring system underlying age-associated impairment in TNF-alpha production in rat macrophages.J Immunol. 1999; 163: 3468-3473PubMed Google Scholar). In this regard, recent reports suggest that DHEA can restore age-associated defects in the PKC signal transduction pathways and related functional responses (Corsini et al., 2002Corsini E. Lucchi L. Meroni M. et al.In vivo dehydroepiandrosterone restores age-associated defects in the protein kinase C signal transduction pathway and related functional responses.J Immunol. 2002; 168: 1753-1758Crossref PubMed Scopus (44) Google Scholar). In our study DHEA globally inhibits pro-inflammatory cytokine production by isolated macrophages. The reduction of IL-6, MIF and TNF-αin vitro closely parallels the observation in wound tissue following DHEA treatment, and the effects of DHEA are again mediated by estrogenic conversion. Intriguingly, this effect appears to be via the ER in the case of MIF, but through non-classical pathways for IL-6 and TNF-α. The PI3 kinase pathway plays a central role in regulating inflammatory cell TNF-α production, and macrophage chemotaxis (Ojaniemi et al., 2003Ojaniemi M. Glumoff V. Harju K. Liljeroos M. Vuori K. Hallman M. Phosphatidylinositol 3-kinase is involved in Toll-like receptor 4-mediated cytokine expression in mouse macrophages.Eur J Immunol. 2003; 33: 597-605Crossref PubMed Scopus (264) Google Scholar; Strassheim et al., 2004Strassheim D. Asehnoune K. Park J.S. et al.Phosphoinositide 3-kinase and Akt occupy central roles in inflammatory responses of Toll-like receptor 2-stimulated neutrophils.J Immunol. 2004; 172: 5727-5733Crossref PubMed Scopus (118) Google Scholar; Weiss-Haljiti et al., 2004Weiss-Haljiti C. Pasquali C. Ji H. et al.Involvement of phosphoinositide 3-kinase gamma, Rac, and PAK signaling in chemokine-induced macrophage migration.J Biol Chem. 2004; 279: 43273-43284Crossref PubMed Scopus (83) Google Scholar). Via non ER/AR mechanisms, DHEA induces both genomic and non-genomic effects involving the ERK1/2 MAP kinase pathway but not the PI3 kinase/Akt pathway, however DHEA may act via PI3 kinase in regulating glucose uptake (Ishizuka et al., 1999Ishizuka T. Kajita K. Miura A. et al.DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.Am J Physiol. 1999; 276: E196-E204PubMed Google Scholar; Simoncini et al., 2003Simoncini T. Mannella P. Fornari L. et al.Dehydroepiandrosterone modulates endothelial nitric oxide synthesis via direct genomic and nongenomic mechanisms.Endocrinology. 2003; 144: 3449-3455Crossref PubMed Scopus (159) Google Scho
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