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Premature Senescence of Balding Dermal Papilla Cells In Vitro Is Associated with p16INK4a Expression

      Androgenetic alopecia (AGA), a hereditary disorder that involves the progressive thinning of hair in a defined pattern, is driven by androgens. The hair follicle dermal papilla (DP) expresses androgen receptors (AR) and plays an important role in the control of normal hair growth. In AGA, it has been proposed that the inhibitory actions of androgens are mediated via the DP although the molecular nature of these interactions is poorly understood. To investigate mechanisms of AGA, we cultured DP cells (DPC) from balding and non-balding scalp and confirmed previous reports that balding DPC grow slower in vitro than non-balding DPC. Loss of proliferative capacity of balding DPC was associated with changes in cell morphology, expression of senescence-associated β-galactosidase, as well as decreased expression of proliferating cell nuclear antigen and Bmi-1; upregulation of p16INK4a/pRb and nuclear expression of markers of oxidative stress and DNA damage including heat shock protein-27, super oxide dismutase catalase, ataxia-telangiectasia-mutated kinase (ATM), and ATM- and Rad3-related protein. Premature senescence of balding DPC in vitro in association with expression of p16INK4a/pRB suggests that balding DPC are sensitive to environmental stress and identifies alternative pathways that could lead to novel therapeutic strategies for treatment of AGA.

      Abbreviations:

      AGA
      androgenetic alopecia
      AR
      androgen receptor
      ATM
      ataxia-telangiectasia mutated kinase
      ATR
      ATM- and Rad3-related protein
      DP
      dermal papilla
      DPC
      DP cell
      HSP-27
      heat shock protein-27
      PBS
      phosphate-buffered saline
      PCNA
      proliferating cell nuclear antigen
      SA-β-Gal
      senescence-associated β-galactosidase
      SOD-1
      superoxide dismutase 1
      TGF-β
      transforming growth factor-β

      Introduction

      It is well known that human hair follicles at specific body sites are responsive to androgens (
      • Randall V.A.
      • Hibberts N.A.
      • Thornton M.J.
      • Hamada K.
      • Merrick A.E.
      • Kato S.
      • et al.
      The hair follicle: a paradoxical androgen target organ.
      ). In beard, axillary and pubic regions, androgens stimulate the conversion of vellus to terminal hairs, whereas in frontal scalp regions of genetically predisposed individuals androgens trigger miniaturization of hair follicles via conversion of terminal to vellus hair follicles (
      • Hamilton J.B.
      Patterned loss of hair in man: types and incidence.
      ). The response of hair follicles to androgens appears to be dependent upon the end-organ sensitivity of individual hair follicles (
      • Deplewski D.
      • Rosenfield R.L.
      Role of hormones in pilosebaceous unit development.
      ). Androgen receptors (ARs) have been detected in the dermal papilla (DP) by ligand-binding assays (
      • Randall V.A.
      • Thornton M.J.
      • Hamada K.
      • Redfern C.P.
      • Nutbrown M.
      • Ebling F.J.
      • et al.
      Androgens and the hair follicle. Cultured human dermal papilla cells as a model system.
      ) and immunohistochemistry (
      • Choudhry R.
      • Hodgins M.B.
      • Van der Kwast T.H.
      • Brinkmann A.O.
      • Boersma W.J.
      Localization of androgen receptors in human skin by immunohistochemistry: implications for the hormonal regulation of hair growth, sebaceous glands and sweat glands.
      ). DP cells (DPC) from androgen-dependent follicles contain more AR than non-androgen-dependent follicles (
      • Randall V.A.
      • Thornton M.J.
      • Hamada K.
      • Messenger A.G.
      Mechanism of androgen action in cultured dermal papilla cells derived from human hair follicles with varying responses to androgens in vivo.
      ;
      • Hibberts N.A.
      • Howell A.E.
      • Randall V.A.
      Balding hair follicle dermal papilla cells contain higher levels of androgen receptors than those from non-balding scalp.
      ) and have higher levels of 5-α reductase activity (
      • Itami S.
      • Kurata S.
      • Takayasu S.
      5 Alpha-reductase activity in cultured human dermal papilla cells from beard compared with reticular dermal fibroblasts.
      ;
      • Thornton M.J.
      • Laing I.
      • Hamada K.
      • Messenger A.G.
      • Randall V.A.
      Differences in testosterone metabolism by beard and scalp hair follicle dermal papilla cells.
      ). The DP plays an important role in controlling hair growth and the hair growth cycle (
      • Oliver R.F.
      Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat.
      ) and secretes a range of growth regulatory factors (
      • Peus D.
      • Pittelkow M.R.
      Growth factors in hair organ development and the hair growth cycle.
      ;
      • Botchkarev V.A.
      • Kishimoto J.
      Molecular control of epithelial–mesenchymal interactions during hair follicle cycling.
      ).
      The molecular mechanisms by which androgens mediate hair growth and especially their mechanism of action in androgenetic alopecia (AGA) are poorly understood. However, models of androgen-mediated regulation of hair growth propose that androgen binding to the AR in the DP effects changes in gene transcription that regulate hair growth and the hair growth cycle (
      • Itami S.
      • Kurata S.
      • Sonoda T.
      • Takayasu S.
      Mechanism of action of androgen in dermal papilla cells.
      ;
      • Randall V.A.
      • Thornton M.J.
      • Hamada K.
      • Redfern C.P.
      • Nutbrown M.
      • Ebling F.J.
      • et al.
      Androgens and the hair follicle. Cultured human dermal papilla cells as a model system.
      ). In support of these models,
      • Itami S.
      • Kurata S.
      • Takayasu S.
      Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor-I from dermal papilla cells.
      have shown that cultured beard DPC secrete IGF-I in response to androgen and
      • Inui S.
      • Fukuzato Y.
      • Nakajima T.
      • Yoshikawa K.
      • Itami S.
      Androgen-inducible TGF-beta1 from balding dermal papilla cells inhibits epithelial cell growth: a clue to understand paradoxical effects of androgen on human hair growth.
      have reported that dihydrotestosterone stimulates secretion of transforming growth factor-β (TGF-β) by cultured balding scalp DP. TGF-β is known to induce a catagen-like morphology and inhibit human hair follicle growth in vitro (
      • Philpott M.P.
      • Green M.R.
      • Kealey T.
      Human hair growth in vitro.
      ) and more recently
      • Hamada K.
      • Randall V.A.
      Inhibitory autocrine factors produced by the mesenchyme-derived hair follicle dermal papilla may be a key to male pattern baldness.
      have shown that balding DPC secrete factors that delay the onset of anagen in mice in vivo. Although the nature of these inhibitory factors remains to be determined, the observations of (
      • Hamada K.
      • Randall V.A.
      Inhibitory autocrine factors produced by the mesenchyme-derived hair follicle dermal papilla may be a key to male pattern baldness.
      ) combined with that of
      • Inui S.
      • Fukuzato Y.
      • Nakajima T.
      • Yoshikawa K.
      • Itami S.
      Androgen-inducible TGF-beta1 from balding dermal papilla cells inhibits epithelial cell growth: a clue to understand paradoxical effects of androgen on human hair growth.
      may explain one mechanism by which the progressive miniaturization of hair follicles in AGA may be driven (
      • Whiting D.A.
      Possible mechanisms of miniaturization during androgenetic alopecia or pattern hair loss.
      ).
      We now confirm previous studies showing that balding DPC have much slower growth rates in vitro when compared with non-balding DPC (
      • Randall V.A.
      • Hibberts N.A.
      • Hamada K.
      A comparison of the culture and growth of dermal papilla cells from hair follicles from non-balding and balding (androgenetic alopecia) scalp.
      ). We demonstrate further that balding but not non-balding DPC undergo premature senescence in vitro and that this is associated with expression of p16INK4a/pRb. In addition, balding DPC expressed a number of markers of oxidative stress and DNA damage, including heat shock protein-27 (HSP-27), super oxide dismutase 1, catalase, ataxia-telangiectasia-mutated kinase (ATM), and the ATM- and Rad3-related protein (ATR). As the p16INK4a/pRb pathway is involved in cell senescence in response to environmental stress, we suggest that the premature senescence of balding DPC in vitro reflects an increased, in vivo, sensitivity of balding DPC to environmental stress.

      Results

      Loss of proliferative capacity in balding DPC is associated with premature cell senescence

      Cell proliferation assays using Alamar Blue (Figure 1) confirm previous data from other groups (
      • Randall V.A.
      • Hibberts N.A.
      • Hamada K.
      A comparison of the culture and growth of dermal papilla cells from hair follicles from non-balding and balding (androgenetic alopecia) scalp.
      ) and show that growth rates of balding DPC in vitro are much slower than of non-balding DPC. In addition to confirming these slower growth rates in balding DPC, we also observed that while non-balding DPC seeded at a density of 2 × 104 cells would undergo repeated cell passage. DPC from balding scalp seeded at the same density (2 × 104 cells) usually underwent growth arrest and could rarely be passaged beyond passage P2–P4. Therefore, it would appear that the slower growth rate of balding DPC seen by
      • Randall V.A.
      • Hibberts N.A.
      • Hamada K.
      A comparison of the culture and growth of dermal papilla cells from hair follicles from non-balding and balding (androgenetic alopecia) scalp.
      and in this study occurs as a result of rapid exhaustion of proliferative ability in vitro and that this does not occur in non-balding DPC of the same passage number.
      Figure thumbnail gr1
      Figure 1Proliferation of balding and non-balding DPC in vitro. Alamar Blue cell proliferation assay for passage 2 balding and non-balding DPCs, showing slower rate of cell proliferation for balding DPC when compared with non-balding cells. Experiments were carried out in triplicate for each time point and represent n=3 separate experiments.
      The loss of proliferative capacity and the ability to passage balding DPC was associated with a change in phenotype from typically elongated fibroblastic cells to much larger flatter, polymorphic cells with very pronounced stress filaments (Figure 2), a characteristic of senescent fibroblasts (
      • Bayreuther K.
      • Rodemann H.P.
      • Hommel R.
      • Dittmann K.
      • Albiez M.
      • Francz P.I.
      Human skin fibroblasts in vitro differentiate along a terminal cell lineage.
      ;
      • Alaluf S.
      • Muir-Howie H.
      • Hu H.L.
      • Evans A.
      • Green M.R.
      Atmospheric oxygen accelerates the induction of a post-mitotic phenotype in human dermal fibroblasts: the key protective role of glutathione.
      ;
      • Chen J.H.
      • Stoeber K.
      • Kingsbury S.
      • Ozanne S.E.
      • Williams G.H.
      • Hales C.N.
      Loss of proliferative capacity and induction of senescence in oxidatively stressed human fibroblasts.
      ). To establish further whether this loss of proliferative capacity was associated with premature senescence in cultured balding DPC, we used a well-characterized marker of in vitro cell senescence namely senescence-associated β-galactosidase (SA-β-Gal) (
      • Dimri G.P.
      • Lee X.
      • Basile G.
      • Acosta M.
      • Scott G.
      • Roskelley C.
      • et al.
      A biomarker that identifies senescent human cells in culture and in aging skin in vivo.
      ). Balding DPC at passage P2 showed strong positive SA-β-Gal staining in the majority of cells, whereas non-balding DPC of the same passage (i.e., P2) were nearly all negative for SA-β-Gal although occasional positive cells were observed (Figure 3a and b). Immunocytochemistry for proliferating cell nuclear antigen (PCNA) revealed a very weak proliferation index in balding DPC when compared with non-balding DPC (Figure 3c and d). Together these data strongly indicate that the loss of proliferative capacity in balding DPC is associated with premature cell senescence. Moreover, the fact that this is not seen in non-balding DPC suggests that this premature senescence is a characteristic of balding cells in vitro.
      Figure thumbnail gr2
      Figure 2Morphology of balding and non-balding DPC in vitro. (a) Balding DPC showing an enlarged, flattened, and senescent-like phenotype at passage 2 compared with (b) non-balding DPC showing normal morphology at passage 4. Images were taken under a light microscope. Bar=100 μm.
      Figure thumbnail gr3
      Figure 3Senescence associated SA-β-Gal and PCNA expression in balding and non-balding DPC. (a) SA-β-Gal staining of balding DPC passage 2 demonstrates increased SA-β-Gal activity when compared with (b) non-balding DPC of the same passage. (d) Expression of nuclear PCNA is seen in non-balding DPC, (c) in contrast there is a marked decrease in PCNA expression in balding cells. Bar=100 μm.

      Senescence of balding DPC is associated with expression of p16INK4a, pRb and absence of Bmi-1

      To strengthen the connection between loss of proliferative capacity and premature senescence in balding DPC, we investigated expression of the cyclin-dependent kinase inhibitor p16INK4a, the retinoblastoma tumor supressor protein pRb, and the tumor supressor protein p53. Because balding DPC are slow growing, it is not possible to generate sufficient number of cells for western blot analysis. We therefore cultured balding and non-balding DPC on glass coverslips and used immunocytochemistry to investigate expression of p16INK4a, pRb, and p53. We observed that balding DPC consistently exhibited increased expression of p16INK4a and pRb (Figure 4a and c), whereas their expression was undetectable in non-balding DPC (Figure 4b and d). Neither balding nor non-balding DPC expressed p53 (data not shown) suggesting that the senescence in balding DPC is mediated via the p16INK4a/Rb pathway and may therefore be associated with cellular stress and not replicative lifespan (
      • Ramirez R.D.
      • Morales C.P.
      • Herbert B.S.
      • Rohde J.M.
      • Passons C.
      • Shay J.W.
      • et al.
      Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions.
      ;
      • Jacobs J.J.
      • de Lange T.
      Significant role for p16INK4a in p53-independent telomere-directed senescence.
      ). In addition, we also examined Bmi-1 expression in balding and non-balding DPC. We show that while Bmi-1 is strongly expressed in the nucleus of non-balding cells, Bmi-1 is downregulated in balding DPC (Figure 4e and f). Expression of Bmi-1 in non-balding DPC and its role as a transcriptional repressor of the INK4a locus correlates with lack of p16INK4a in these cells, whereas in balding DPC lack of Bmi-1 expression is concomitant with upregulation of p16INK4a expression and further supports our hypothesis that premature senescence of balding DPC is associated with the p16INK4a pathway.
      Figure thumbnail gr4
      Figure 4Expression of p16, pRB, and Bmi-1 in balding and non-balding DPC. (a and b) Immunocytochemistry for p16, (c and d) pRB, and (e and f) Bmi-1 in balding and non-balding DPC show expression of p16 and pRb in (a and c) balding but not in (b and d) non-balding DPC. Bmi-1 a negative regulator of p16 is downregulated in (e) balding but strongly expressed in (f) the nucleus of non-balding DPC. Bar=100 μm.

      Balding DPC show changes in levels of expression and distribution of stress-response proteins

      Because balding DPC expressed p16INK4a and pRb but not p53, we hypothesized that premature senescence of balding DPC may be associated with DNA damage or oxidative stress, which are known to cause fibroblast senescence in vitro (
      • Parrinello S.
      • Samper E.
      • Krtolica A.
      • Goldstein J.
      • Melov S.
      • Campisi J.
      Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.
      ;
      • Chen J.H.
      • Stoeber K.
      • Kingsbury S.
      • Ozanne S.E.
      • Williams G.H.
      • Hales C.N.
      Loss of proliferative capacity and induction of senescence in oxidatively stressed human fibroblasts.
      ). By immunocytochemistry, we investigated expression of the stress-response HSP-27, catalase, and superoxide dismutase (SOD-1) as well as the DNA damage proteins ATM and ATR. Expressions of catalase (Figure 5a and b) and SOD-1 (Figure 5c and d) were detected at low levels in non-balding DPC, where their expression was predominantly cytoplasmic. Strikingly in balding DPC, expression of both catalase and SOD-1 was restricted to nuclear and perinuclear compartments and the cytoplasm was mainly negative. We observed that HSP-27 was elevated in balding compared with non-balding DPC (Figure 5e and f). Both DNA damage proteins ATM and ATR were detected in balding but not in non-balding DPC (Figure 6), which suggests that DNA damage possibly resulting from oxidative stress may be involved in the premature senescence of balding DPC in vitro.
      Figure thumbnail gr5
      Figure 5Expression of catalase, SOD-1, and HSP-27 in balding and non-balding DPC. Balding DPC showed a strong nuclear (a) catalase and (c) SOD-1 expression, which contrasted to the mainly cytoplasmic expression in (b and d) non-balding DPC. Increased expression of HSP-27 was also observed in (e) balding but not in (f) non-balding DPC. Bar=100 μm.
      Figure thumbnail gr6
      Figure 6Expression of DNA damage proteins in balding and non-balding DPC. Increased expression of activated form of (a) ATM and (c) ATR is observed in balding but not in (b and d) non-balding DPCs. Bar=100 μm.

      Discussion

      Previous studies have reported that DPC from balding scalp grow slower in vitro than DPC from non-balding scalp (
      • Randall V.A.
      • Hibberts N.A.
      • Hamada K.
      A comparison of the culture and growth of dermal papilla cells from hair follicles from non-balding and balding (androgenetic alopecia) scalp.
      ). In this study, we were able to confirm the slow growing nature of balding DPC in comparison to non-balding cells. However, we show that this loss of proliferative capacity as demonstrated by both cell proliferation assay and decreased PCNA expression is associated with the onset of premature senescence as characterized by the appearance of flat, enlarged and vacuolated cells, SA-β-Gal staining, and increased_expression of p16INK4a and pRb (
      • Bayreuther K.
      • Rodemann H.P.
      • Hommel R.
      • Dittmann K.
      • Albiez M.
      • Francz P.I.
      Human skin fibroblasts in vitro differentiate along a terminal cell lineage.
      ;
      • Alaluf S.
      • Muir-Howie H.
      • Hu H.L.
      • Evans A.
      • Green M.R.
      Atmospheric oxygen accelerates the induction of a post-mitotic phenotype in human dermal fibroblasts: the key protective role of glutathione.
      ;
      • Chen J.H.
      • Stoeber K.
      • Kingsbury S.
      • Ozanne S.E.
      • Williams G.H.
      • Hales C.N.
      Loss of proliferative capacity and induction of senescence in oxidatively stressed human fibroblasts.
      ).
      A number of physiological stimuli can induce cell senescence. These include extensive passage in culture commonly referred to as replicative senescence (
      • Hayflick L.
      • Moorhead P.S.
      The serial cultivation of human diploid cell strains.
      ), exposure to environmental stress (
      • Chen Q.
      • Fischer A.
      • Reagan J.D.
      • Yan L.J.
      • Ames B.N.
      Oxidative DNA damage and senescence of human diploid fibroblast cells.
      ;
      • von Zglinicki T.
      • Saretzki G.
      • Docke W.
      • Lotze C.
      Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence?.
      ), and oncogene activation (
      • Serrano M.
      • Lin A.W.
      • McCurrach M.E.
      • Beach D.
      • Lowe S.W.
      Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.
      ;
      • de Stanchina E.
      • McCurrach M.E.
      • Zindy F.
      • Shieh S.Y.
      • Ferbeyre G.
      • Samuelson A.V.
      • et al.
      E1A signaling to p53 involves the p19(ARF) tumor suppressor.
      ). Replicative senescence following extensive passage in culture is usually associated with progressive telomere shortening and involves the activation of p53 and p21Cip1 (
      • Dulic V.
      • Beney G.E.
      • Frebourg G.
      • Drullinger L.F.
      • Stein G.H.
      Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts.
      ;
      • Itahana K.
      • Dimri G.
      • Campisi J.
      Regulation of cellular senescence by p53.
      ). Whereas senescence induced by environmental stress is mediated via the p16INK4a/Rb pathway (
      • Jacobs J.J.
      • de Lange T.
      Significant role for p16INK4a in p53-independent telomere-directed senescence.
      ). Our data showing that senescence of balding DPC is associated with increased expression of p16INK4a and pRb and not p53 suggests that the senescence of balding DPC in vitro is stimulated by environmental stress and is not due to replicative senescence. It is known that the rapid onset of senescence in some cell types may be caused by the stress of cell culture including high oxygen tension, lack of interactions with neighboring cells, growth on plastic as well as growth factor, and nutrient deficiencies of tissue culture medium (
      • Sherr C.J.
      • DePinho R.A.
      Cellular senescence: mitotic clock or culture shock?.
      ;
      • Ben-Porath I.
      • Weinberg R.A.
      The signals and pathways activating cellular senescence.
      ). In our study, only balding DPC showed this premature stress response with non-balding DPC undergoing repeated passage in vitro. This suggests that balding DPC are more susceptible to the stress of tissue culture than non-balding cells. That balding DPC may be more susceptible to stress in general was further highlighted by our observation that when matched cultures of balding and non-balding DPC from the same patients and same passage (P2) were stored in liquid nitrogen; non-balding DPC could be routinely recovered back into culture, whereas balding DPC could not be retrieved (unpublished observation). In total, we have cultured balding DPC from over 20 patients and all have been slow to grow in culture and shown premature senescence. In contrast, we have never seen this in a similar number of non-balding DPC cultures. Furthermore, we routinely culture DPC from female facelift skin, pubic and hirsute biopsies as well as male beard and have not seen premature senescence in any of these cultures. Although we cannot completely exclude the possibility that this premature senescence is a site-specific phenomenon of frontal scalp follicles, we suggest this is unlikely for the reasons outlined above. It has not been possible to obtain frontal scalp biopsies from healthy male patients, although we have obtained non-balding male scalp samples from plastic surgery and DPC from these follicles did not senesce.
      Overexpression of Bmi-1, a suppressor of p16 and p19, extends the replicative lifespan of mouse and human fibroblasts (
      • Jacobs J.J.
      • Kieboom K.
      • Marino S.
      • DePinho R.A.
      • van Lohuizen M.
      The oncogene and polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus.
      ). Therefore, our data showing Bmi-1 expression in non-balding but not in balding DPC correlates with the respective expression of p16INK4a in these cells and provides further evidence that the premature senescence of balding DPC is associated with the p16INK4a pathway and that this may be mediated via downregulation of Bmi-1.
      Increased expression of p16INK4a has been reported previously in cell senescence resulting from DNA damage (
      • Robles S.J.
      • Adami G.R.
      Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts.
      ) and oxidative stress (
      • Chen Q.M.
      Replicative senescence and oxidant-induced premature senescence. Beyond the control of cell cycle checkpoints.
      ). The phosphatidylinositol kinase-related kinases, ATM and ATR have been shown to be essential components of the machinery that controls checkpoint activation in response to DNA damage (
      • Zhou B.B.
      • Elledge S.J.
      The DNA damage response: putting checkpoints in perspective.
      ). Therefore, our observation that the expression of activated form of ATM and ATR were only detected in balding and not in the non-balding DPC suggests that DNA damage may play an important role in the onset of premature senescence in balding DPC. However, whether this is due to oxidative stress or other mechanisms remains to be determined.
      SOD-1 and catalase prevent or terminate the reactions of reactive oxygen species. SOD-1 and catalase were observed in both balding and non-balding DPC. However, marked differences in location were observed with both SOD-1 and catalase being mainly cytoplasmic in non-balding DP, whereas in balding DPC expression was mainly perinuclear/nuclear. This expression pattern was persistent in all the balding DPC studied. SOD-1 is normally expressed in the mitochondria and cytoplasm of eukaryotic cells, whereas catalase is normally present in the peroxisome fraction. Overexpression of catalase in cytosolic or mitochondrial compartments has been shown to protect cells against oxidative injury (
      • Bai J.
      • Rodriguez A.M.
      • Melendez J.A.
      • Cederbaum A.I.
      Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury.
      ). However, catalase has also been reported in the nuclear matrix of guinea-pig hepatocytes where it is believed to play a similar protective role (
      • Yamamoto K.
      • Volkl A.
      • Hashimoto T.
      • Fahimi H.D.
      Catalase in guinea pig hepatocytes is localized in cytoplasm, nuclear matrix and peroxisomes.
      ). Therefore, in balding DPC, the nuclear localization of catalase and SOD-1 may reflect an attempt by the cells to protect its nucleus from oxidative damage. Finally, our data showing that HSP-27 was only detected in balding DPC and not in non-balding cells also suggests that balding cells are more sensitive to environmental stress (
      • Takayama S.
      • Reed J.C.
      • Homma S.
      Heat-shock proteins as regulators of apoptosis.
      ).
      • Inui S.
      • Fukuzato Y.
      • Nakajima T.
      • Yoshikawa K.
      • Itami S.
      Androgen-inducible TGF-beta1 from balding dermal papilla cells inhibits epithelial cell growth: a clue to understand paradoxical effects of androgen on human hair growth.
      have reported that cultured balding DPC secrete more TGF-β than non-balding cells. TGF-β is known to stimulate oxidative stress in human fibroblasts (
      • Thannickal V.J.
      • Fanburg B.L.
      Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1.
      ). It is therefore possible that increased production of TGF-β in vivo in response to androgens may initiate cellular stress responses in balding hair follicles in vivo. Senescent cells are known to have altered patterns of gene expression and secrete a range of growth factors, extracellular matrix proteins, and degradative enzymes (
      • Bavik C.
      • Coleman I.
      • Dean J.P.
      • Knudsen B.
      • Plymate S.
      • Nelson P.S.
      The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms.
      ). These may have a negative impact on hair growth as recently demonstrated by
      • Hamada K.
      • Randall V.A.
      Inhibitory autocrine factors produced by the mesenchyme-derived hair follicle dermal papilla may be a key to male pattern baldness.
      who showed that cultured balding DPC secreted factors that inhibited anagen onset in mice.
      In conclusion, we demonstrate that the proliferative lifespan of DPC from the balding patients and cultured under normal cell culture conditions is severely limited and associated with stress-induced cell senescence. Cellular senescence is known to represent an aging-associated process and because senescent cells are known to accumulate with age (
      • Dimri G.P.
      • Lee X.
      • Basile G.
      • Acosta M.
      • Scott G.
      • Roskelley C.
      • et al.
      A biomarker that identifies senescent human cells in culture and in aging skin in vivo.
      ), it is possible that in some scalp follicles aging results in increased susceptibility to senescence. We suggest therefore that the premature stress-induced senescence seen in vitro reflects an increased susceptibility of balding DP to stress in vivo. Even if cell senescence does not directly contribute to AGA, our data clearly demonstrate that balding DPC are more responsive to environmental stress than non-balding DPC and even subtle changes in environment may well contribute to a decrease in function of the DP in vivo leading to miniaturization of hair follicles. Therefore, mechanisms that target stress pathways may provide additional therapies for the treatment of AGA.

      Materials and Methods

      Isolation and culture of human DPC

      Matched punch biopsies (2 mm) were taken from balding (frontal) and non-balding (occipital) scalps of male patients who were undergoing hair transplant surgery for male-pattern baldness. The ethics approval for this study was obtained from East London and City health authority (T/98/008) and all biopsies were taken with full patient consent. Furthermore, all experiments adhered to the Declaration of Helsinki Principles. The samples used in these studies were taken from patients not currently using any hair loss medications. Hair follicles were microdissected from balding and non-balding scalp biopsies and the DP isolated under a dissecting microscope as described previously (
      • Philpott M.P.
      • Sanders D.
      • Westgate G.E.
      • Kealey T.
      Human hair growth in vitro: a model for the study of hair follicle biology.
      ). We were able to isolate between one and three balding and non-balding DPs from each 2 mm biopsy. These were then cultured in the DP media containing Williams E media supplemented with 15% (v/v) fetal bovine serum, 2 mM glutamine, 100 ng ml−1 hydrocortisone, 10 μg ml−1 insulin, 100 U ml−1 penicillin G, and 100 mg ml−1 streptomycin (all culture reagents supplied by Sigma, Dorset, UK) at 37°C in an atmosphere of 5% CO2 and 90% air (
      • Messenger A.G.
      The culture of dermal papilla cells from human hair follicles.
      ).

      Alamar Blue™ cell proliferation assay

      The proliferation rates of the balding and non-balding DPC at passage 2 were measured using the Alamar Blue™ cell proliferation assay (Biosource International Inc.) according to the manufacturer's instructions (
      • Nociari M.M.
      • Shalev A.
      • Benias P.
      • Russo C.
      A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity.
      ). In brief, 2 × 104 cells were seeded in individual wells of six-well culture plates and maintained over 10 days. Proliferation assays were carried out on days 1, 3, 5, 7, and 10 by adding Alamar Blue™ reagent directly to the culture medium and incubating for 6 hours at 37°C. Fluorescence was measured with excitation wavelength at 530–560 nm and emission 590 nm using a Wallac plate reader.

      SA-β-Gal assay

      Cytochemical detection of β-galactosidase was carried out according to the method of
      • Dimri G.P.
      • Lee X.
      • Basile G.
      • Acosta M.
      • Scott G.
      • Roskelley C.
      • et al.
      A biomarker that identifies senescent human cells in culture and in aging skin in vivo.
      . Briefly, monolayers of cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 4% (v/v) paraformaldehyde for 15 minutes at room temperature. The cells were then washed twice with PBS and then incubated overnight at 37°C with 1 mg ml−1 5-bromo-4-chloro-3-inolyl-b-D-galactoside in dimethylformamide (20 mg ml−1 stock), 5 mm potassium ferrocyanide, 5 mm potassium ferricyanide, 150 mm NaCl, 2 mm MgCl2, and 40 mm citric acid/sodium phosphate, pH 6.0.

      Immunocytochemistry

      Balding and non-balding DPCs were seeded at a density of 1 × 105 on to flame sterilized glass coverslips that had been placed in six-well plates and cultured. The cells were then fixed with 4% (v/v) paraformaldehyde, permeabilized with 0.1% (v/v) Triton, and their endogenous peroxidase blocked in 0.3% (v/v) hydrogen peroxide/70% methanol (v/v) followed by incubation in PBS-diluted horse serum for 10 minutes at room temperature. Coverslips were incubated with primary antibody diluted in PBS overnight at 4°C. Antibodies used were as follows: PCNA (ab29; Abcam, Cambridge, UK), p53 (ab7757; Abcam), p21 (H-164; Santa Cruz, Wiltshire, UK), p16 (H-156; Santa Cruz), (G3–245; BD Pharmingen, Oxford, UK), catalase (ab1877; Abcam), SOD-1 (FL-154; Santa Cruz), and HSP-27 (H-77, Santa Cruz). After incubation with primary antibodies, cells were washed three times with PBS. Secondary and tertiary antibodies from Vectastain ABC Universal Elite kits (Vector Laboratories, Peterborough, UK) were used in accordance with the manufacturer's instructions. Immunolocalization was visualized with 3,3′-diaminobenzidine tetrahydrochloride solution (Vectastain, Southgate, UK), cells were counterstained with hematoxylin and mounted in DePeX (BDH Laboratory Supplies, Poole, UK).

      Conflict of Interest

      The authors state no conflict of interest.

      ACKNOWLEDGMENTS

      We are grateful to Dr Teck Teh for his technical assistance.

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