Induction of transforming growth factor-beta 1 by androgen is mediated by reactive oxygen species in hair follicle dermal papilla cells.

The progression of androgenetic alopecia is closely related to androgen-inducible transforming growth factor (TGF)-ß1 secretion by hair follicle dermal papilla cells (DPCs) in bald scalp. Physiological levels of androgen exposure were reported to increase reactive oxygen species (ROS) generation. In this study, rat vibrissae dermal papilla cells (DP-6) transfected with androgen receptor showed increased ROS production following androgen treatment. We confirmed that TGF-ß1 secretion is increased by androgen treatment in DP-6, whereas androgen-inducible TGF-ß1 was significantly suppressed by the ROS-scavenger, N-acetyl cysteine. Therefore, we suggest that induction of TGF-ß1 by androgen is mediated by ROS in hair follicle DPCs.

Comparative secretome analysis of human follicular dermal papilla cells and fibroblasts using shotgun proteomics.

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically- validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.

Photoaging-associated changes in epidermal proliferative cell fractions in vivo.

The epidermis is a dynamic epithelium with constant renewal throughout life. Epidermal homeostasis depends on two types of proliferative cells, keratinocyte stem cells (KSCs), and transit amplifying (TA) cells. In the case of chronologic aging, levels of KSCs tend to decrease and change functionally. However, little is known about the effect of photoaging on epidermal proliferative subtype populations. The aim of this study was to validate involucrin/beta1-integrin ratio as a molecular marker of epidermal photoaging, and to investigate the effects of photoaging caused by chronic UV exposure on the proliferative subtype populations. A total of 15 male volunteers (age range 20-24 and 77-85 years, Fitzpatrick skin phototype III-IV) provided sun-exposed and sun-protected skin samples for real-time RT-PCR, Western blot analysis and immunostaining. Fractional changes in proliferative subtype populations in photoaged and chronologically aged skins were analyzed by flow cytometry. The expression of beta1-integrin was found to be significantly reduced in photoaged skin and ratios of the expressions of involucrin to beta1-integrin were increased 2.6-fold only in elderly subjects. Interestingly, immunostaining of the sun-exposed skins of elderly subjects showed aberrant beta1-integrin expression over the basal layer and greater numbers of Ki-67-positive cells than in sun-protected buttock skin. Flow cytometric analysis revealed that the proportion of KSCs to TA cells was reversed in sun-exposed and sun-protected skins of elderly subjects. Our results suggest that KSC numbers may be lower in photoaged skin than in chronologically aged skin and could be applied to hyperplastic pattern of photoaging. These findings suggest that the epidermis of photoaged skin is impaired in terms of its proliferative potential by attempting to repair chronic UV exposure and that photoaging may be associated with alteration in the two proliferative cell fractions.

The effect of tripeptide-copper complex on human hair growth in vitro.

The tripeptide-copper complex, described as a growth factor for various kinds of differentiated cells, stimulates the proliferation of dermal fibroblasts and elevates the production of vascular endothelial growth factor, but decreased the secretion of transforming growth factor-beta1 by dermal fibroblasts. Dermal papilla cells (DPCs) are specialized fibroblasts, which are important in the morphogenesis and growth of hair follicles. In the present study, the effects of L-alanyl-L-histidyl-L-lysine-Cu2+ (AHK-Cu) on human hair growth ex vivo and cultured dermal papilla cells were evaluated. AHK-Cu (10(-12) - 10(-9) M) stimulated the elongation of human hair follicles ex vivo and the proliferation of DPCs in vitro. Annexin V-fluorescein isothiocyanate/propidium iodide labeling and flow cytometric analysis showed that 10(-9) M AHK-Cu reduced the number of apoptotic DPCs, but this decrease was not statistically significant. The ratio of Bcl-2/Bax was elevated, and the levels of the cleaved forms of caspase-3 and PARP were reduced by treatment with 10(-9) M AHK-Cu. The present study proposed that AHK-Cu promotes the growth of human hair follicles, and this stimulatory effect may occur due to stimulation of the proliferation and the preclusion of the apoptosis of DPCs.

The additive effects of minoxidil and retinol on human hair growth in vitro.

Minoxidil enhances hair growth by prolonging the anagen phase and induces new hair growth in androgenetic alopecia (AGA), whereas retinol significantly improves scalp skin condition and promotes hair growth. We investigated the combined effects of minoxidil and retinol on human hair growth in vitro and on cultured human dermal papilla cells (DPCs) and epidermal keratinocytes (HaCaT). The combination of minoxidil and retinol additively promoted hair growth in hair follicle organ cultures. In addition, minoxidil plus retinol more effectively elevated phosphorylated Erk, phosphorylated Akt levels, and the Bcl-2/Bax ratio than minoxidil alone in DPCs and HaCaT. We found that the significant hair shaft elongation demonstrated after minoxidil plus retinol treatment would depend on the dual kinetics associated with the activations of Erk- and Akt-dependent pathways and the prevention of apoptosis by increasing the Bcl-2/Bax ratio.

Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells.

Epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. This study was aimed to investigate the possible mechanisms of EGCG-mediated inhibition against apoptosis in rat pheochromocytoma PC12 cells by exposure to CoCl(2). Exposure to CoCl(2) caused the generation of ROS and induced cell death with appearance of apoptotic morphology and DNA fragmentation. However, EGCG rescued the loss of viability in the cells exposed to CoCl(2) and led the reduction of DNA fragmentation and sub-G(1) fraction of cell cycle. Also, EGCG attenuated the CoCl(2)-induced disruption of mitochondrial membrane potential (DeltaPsim), release of cytochrome c from the mitochondria to cytosol and abolished the CoCl(2)-stimulated activities of the caspase cascades, caspase-9 and caspase-3. In addition, EGCG ameliorated the increase in the Bax to Bcl-2 ratio, a marker of apoptosis proceeding, induced by CoCl(2) treatment. Taken together, the present results suggest that EGCG inhibit the CoCl(2)-induced apoptosis of PC12 cells through the mitochondria-mediated apoptosis pathway involved in modulating the Bcl-2 family.

Expression of androgen and estrogen receptors in human scalp mesenchymal cells in vitro.

The expression levels of sex hormone receptors were identified to be different in human mesenchymal cells [dermal papilla cell (DPC), dermal sheath cell (DSC), dermal fibroblast and (DF)] from occipital scalps. Transcriptional and translational activities of androgen receptor (AR) and estrogen receptor beta (ERbeta) were most intensely expressed in DPC, followed by DSC and DF. On the contrary, estrogen receptor alpha (ERalpha) was shown with the strongest positivity in DSC, succeeded by DPC and DF subsequently. Immunocytochemical staining showed the similar expression to previous patterns. Our results suggest that the expression levels of ER subtypes and AR may be important for the regulation of follicular mesenchymal cells in human scalp. Further studies of the interactions of hormones and receptors in human hair follicles are required to promote our understanding of the effects of sex hormones on hair biology.

Perifollicular fibrosis: pathogenetic role in androgenetic alopecia.

Androgenetic alopecia (AGA) is a dihydrotestosterone (DHT)-mediated process, characterized by continuous miniaturization of androgen reactive hair follicles and accompanied by perifollicular fibrosis of follicular units in histological examination. Testosterone (T: 10(-9)-10(-7) M) treatment increased the expression of type I procollagen at mRNA and protein level. Pretreatment of finasteride (10(-8) M) inhibited the T-induced type I procollagen expression at mRNA (40.2%) and protein levels (24.9%). T treatment increased the expression of transforming growth factor-beta 1 (TGF-beta1) at protein levels by 81.9% in the human scalp dermal fibroblasts (DFs). Pretreatment of finasteride decreased the expression of TGF-beta1 protein induced by an average of T (30.4%). The type I procollagen expression after pretreatment of neutralizing TGF-beta1 antibody (10 microg/ml) was inhibited by an average of 54.3%. Our findings suggest that T-induced TGF-beta1 and type I procollagen expression may contribute to the development of perifollicular fibrosis in the AGA, and the inhibitory effects on T-induced procollagen and TGF-beta1 expression may explain another possible mechanism how finasteride works in AGA.

Jun activation domain-binding protein 1 is required for mitotic checkpoint activation via its involvement in hyperphosphorylation of 53BP1.

PURPOSE: p53-binding protein 1 (53BP1), a participant in the DNA damage response pathway, has also been implicated in the cellular response to mitotic stress conditions. Here, we sought to broaden our understanding of the protein network surrounding 53BP1 by identifying and characterizing a 53BP1-interacting protein. METHOD: Yeast two-hybrid screening was performed to identify possible binding partners of 53BP1. To investigate the functional meaning of the interaction, knock-down cells were established by introduction of antisense construct or siRNA into HeLa cells. The hyperphosphorylation of 53BP1 after treatment with nocodazole, a microtubule-interfering agent, was monitored by immunoblotting. And the cell cycle arrest at mitotic phase was measured by flow cytometry after staining with phospho-(Ser10)-histone H3 antibody. RESULTS: Jun activation domain-binding protein 1 (Jab1) was identified as a 53BP1-binding protein, and the interaction between them was confirmed to occur in mammalian cells. We found that nocodazole-induced 53BP1 hyperphosphorylation was abolished in Jab1 knock-down cells. In addition, ectopic overexpression of Jab1 augmented 53BP1 hyperphosphorylation. On the cellular level, Jab1 knock-down cells exhibited reduced mitotic arrest upon exposure to nocodazole, resulting in cellular resistance to the drug. CONCLUSION: Taken together, these results suggest that Jab1 is required for the hyperphosphorylation of 53BP1 upon mitotic stress conditions and is involved in proper activation of mitotic checkpoint mechanism. Our study also suggests the possibility that modulation of Jab1 activity may be an intriguing approach for enhancing the efficacy of microtubule-interfering anticancer drugs.

Antisense Deoxyoligonucleotides Inhibit Activities of Matrix Metalloproteinase-2 in Human Fibrosarcoma HT1080 Cells.

PURPOSE: MMP-2, 72 kDa-type IV collagenase, plays a major role in the migration and growth of tumor cells, a process that requires the disintegration of basement membrane. Activation of MMP-2 is correlated with the invasiveness of various tumors. The aim of this study was to determine the sequence-specific phosphorothioated oligodeoxynucleotides (ODNs) inhibiting the translation of MMP-2 mRNA and the subsequent invasiveness of tumor cells. MATERIALS AND METHODS: Eight types of antisense ODNs were designed and each (8micro gram/ml) were transfected into HT1080 cells. The effects of these antisense ODNs on MMP expression were examined by gelatin zymography, Western blot, Northern blot and matrigel assay. RESULTS: Antisense-5 (+904~923), antisense-6 (+1274~+1293) and antisense-7 (+1646~+1665) reduced the MMP-2 activity of the culture supernatant in HT1080 fibrosarcoma cells. Treatment with antisense-6 showed inhibition of MMP-2 mRNA and protein, and in vitro invasion in a dose-dependent manner. CONCLUSION: Antisense-6 might be one of the therapeutic candidates for tumor invasion and metastasis.