Extracellular matrix derived from hair and skin fibroblasts stimulates human skin melanocyte tyrosinase activity

There is indirect evidence that both skin and hair melanocytes are regulated by the activity of adjacent cells. In hair, the specialized fibroblasts (dermal papilla cells) appear to play a role in the regulation of hair growth. Hair pigmentation may relate to hair growth. In skin, melanocytes are located adjacent to the basement membrane zone. As far as we are aware, direct interactions of fibroblasts with melanocytes have not previously been investigated. Accordingly, the objective of this study was to develop co-culture conditions in which to investigate whether dermal fibroblasts from skin or hair could influence melanocyte differentiation. The influence of fibroblast-conditioned media, co-culture with fibroblasts, and fibroblast-derived extracellular matrix (ECM) on normal human skin melanocyte tyrosinase activity was examined. Fibroblasts from both skin and hair were capable of altering melanocyte morphology and significantly increasing tyrosinase activity when melanocytes were cultured in the absence, but not the presence, of the major proliferative drives. Although stimulation of tyrosinase activity was detectable with conditioned medium and co-culture with fibroblasts, the most striking result was obtained with the fibroblast-produced ECM which, on average, produced a four-fold increase in tyrosinase activity within 6 days. Thus, the study describes co-culture conditions in which the stimulatory effect of the fibroblast on melanocyte differentiation can be examined.     

Elevated matrix metalloproteinase-2 and -3 production from human diabetic dermal fibroblasts

BACKGROUND: Diabetic foot ulcers are characterized by elevated levels of matrix metalloproteinases (MMP), which could lead to excessive matrix breakdown and disruption to healing. It is unknown if this elevation is a function of wound healing, or if it is present within normal skin and a primary contributor to the increased risk of impaired healing. OBJECTIVES: To determine whether diabetic fibroblasts from unwounded skin show elevated MMP production compared with their nondiabetic counterparts. PATIENTS AND METHODS: Circular skin biopsies (4 mm diameter) were taken from the inside upper arm of four controls without diabetes and from four subjects with insulin-treated diabetes. Fibroblasts were incubated for a further 72 h and conditioned medium was collected and stored at -20 degrees C. The conditioned medium was assessed by gelatin zymography and Western blotting for MMP-2 and MMP-3. RESULTS: Diabetic dermal fibroblasts showed significantly elevated production of MMP-2 (P < 0.05) and pro-MMP-3 (P < 0.05) when compared with their nondiabetic counterparts. CONCLUSIONS: Dermal fibroblasts from normal unwounded skin are characterized by increased MMP production and this may be a primary contributing factor to the increased risk of nonhealing foot ulceration in diabetes.     

SIRT1 modulates expression of matrix metalloproteinases in human dermal fibroblasts

Background SIRT1, an NAD⁺‐dependent histone/protein deacetylase, controls a broad range of cellular functions. Objectives We examined if SIRT1 is involved in the regulation of matrix metalloproteinase (MMP) expression in human dermal fibroblasts. Methods We studied the effect of inhibition of SIRT1 by specific inhibitor and small interfering RNA (siRNA) on MMP‐1 and MMP‐3 expression in human dermal fibroblasts. Results Treatment with a potent and selective inhibitor of SIRT1, EX‐527, increased the basal expression levels of MMP‐1 and MMP‐3 proteins. Knockdown of endogenous SIRT1 by siRNA led to increased expression of MMP‐1 and MMP‐3 at both mRNA and protein levels. SIRT1 knockdown also upregulated MMP protein induction caused by an inflammatory cytokine, interleukin (IL)‐1β. Moreover, treatment with a SIRT1 activator, resveratrol, significantly suppressed IL‐1β‐mediated induction of MMP‐1, which was attenuated by pretreatment with EX‐527. Finally, MMP‐1 promoter activity was increased by EX‐527 in cells treated with or without IL‐1β. Conclusions Our findings suggest that SIRT1 exerts a negative regulatory role in the production of MMP‐1 and MMP‐3 in human dermal fibroblasts.     

Sphingosylphosphorylcholine stimulates cellular fibronectin expression through upregulation of IL-6 in cultured human dermal fibroblasts

Sphingosylphosphorylcholine (SPC) has been reported to stimulate wound healing by its potent mitogenic effect. Fibronectin (FN) is a cell-adhesion protein that plays an important role in cell migration and collagen deposition during wound healing. In order to elucidate further the mechanism involved in the accelerated wound healing stimulated by SPC, we studied the role of SPC in FN production in cultured human dermal fibroblasts. We demonstrated that SPC dose- and time-dependently enhanced the expression of FN in human dermal fibroblast at the protein and mRNA levels. IL-6 is known to stimulate the production of FN in fibroblasts. SPC also markedly induced IL-6 production in cultured human dermal fibroblasts in a dose- and time-dependent manner. We also demonstrated that FN mRNA expression in human dermal fibroblasts was upregulated 4 h after IL-6 treatment. Moreover, pretreatment with neutralizing anti-IL-6 antibodies partially blocked the upregulation of FN mRNA expression induced by SPC in human dermal fibroblasts. These results indicate that SPC may stimulate FN synthesis through IL-6 production in cultured human dermal fibroblasts.     

Propionibacterium acnes stimulates pro-matrix metalloproteinase-2 expression through tumor necrosis factor-alpha in human dermal fibroblasts

Propionibacterium acnes (P. acnes) is a commensal microorganism found in sebum-rich skin and plays a role in acne inflammation by stimulating keratinocyte to produce a number of proinflammatory cytokines. However, the role of P. acnes in the dermis of acne lesions, where tissue remodeling after inflammation eventually takes place, is not known. In this study, we investigated whether P. acnes induces matrix metalloproteinase (MMP), a key enzyme involved in matrix remodeling in human dermal fibroblasts (hDF). We found that P. acnes increased expression of pro-matrix metalloproteinase (proMMP)-2 mRNA/protein in hDF, but not that of proMMP-9. Concomitantly, P. acnes induced tumor necrosis factor-alpha (TNF-alpha) mRNA/protein expression in hDF, which in turn increases both proMMP-2 mRNA and protein expression. P. acnes induced such changes through the activated NF-kappaB pathway. Doxycycline was found to inhibit the expression of proMMP-2 induced either by P. acnes or TNF-alpha. These results suggest that P. acnes stimulates hDF to produce TNF-alpha, which mediates the expression of proMMP-2 through the NF-kappaB pathway. The secretion of proMMP-2 from hDF upon P. acnes stimulation may contribute to the pathogenesis of tissue remodeling in acne skin.     

All-trans retinoic acid stimulates growth and extracellular matrix production in growth-inhibited cultured human skin fibroblasts

All-trans retinoic acid was examined for effects on human dermal fibroblast proliferation and for effects on fibroblast production and expression of non-collagenous and collagenous components of the extracellular matrix in vitro. Fibroblast proliferation was blocked when the cells were cultured in the presence of a serum-free culture medium containing epidermal growth factor, hydrocortisone, insulin, ethanolamine, phosphoethanolamine, and bovine pituitary extract as growth supplements and 0.15 mM Ca++. This level of extracellular Ca++ is lower than that needed to support fibroblast growth. Under these conditions, growth was stimulated by all-trans retinoic acid. Proliferation was also stimulated in the same basal medium without the growth supplements. Growth-promoting concentrations of all-trans retinoic acid ranged from 0.5-2.0 micrograms/ml (1.7-6.6 X 10(-6) M). Stimulation of proliferation was not seen at higher or lower concentrations. Concentrations of all-trans retinoic acid that stimulated proliferation also induced increased production of fibronectin as indicated by biosynthetic labeling/immunoprecipitation and by enzyme-linked immunosorbent assay. Increased production was associated with increased staining for fibronectin in the extracellular matrix. Increased production of two other non-collagenous extracellular matrix component, i.e., thrombospondin and laminin, also occurred in all-trans retinoic acid-treated cells. At 0.5 micrograms/ml, all-trans retinoic acid also stimulated production of type I collagen by the dermal fibroblasts, but at higher concentrations (2.5 micrograms/ml) production of type I collagen was inhibited. These data indicate that all-trans retinoic acid can induce changes in dermal fibroblasts in vitro (i.e., increased proliferation and extracellular matrix production) that mimic the major changes seen in the dermis after topical treatment with this agent.     

Human eosinophils stimulate DNA synthesis and matrix production in dermal fibroblasts

Fibrosis is frequently found in diseases exhibiting tissue eosinophilia, such as some parasitic worm infections, eosinophilia-myalgia syndrome, and eosinophilic fasciitis. Previously, eosinophil extracts have been shown to induce proliferation in neonatal foreskin fibroblasts in vitro. To determine if living eosinophils can induce synthesis of DNA and components of the extracellular matrix in dermal fibroblasts, we cultured purified human eosinophils for 2 or 7 days in the presence of the eosinophil-active cytokines granulocyte/macrophage colony stimulating factor, interleukin-3, or interleukin-5, and added eosinophil-conditioned medium to cultures of dermal fibroblasts. Using flow cytometry, we found that eosinophil-conditioned medium increased by two-fold the percentage of fibroblasts in S-phase. This stimulation of fibroblast DNA synthesis was corroborated using a standard tritiated thymidine assay and the two methods were shown to correlate well with each other. Eosinophil-conditioned medium stimulation of DNA synthesis was dose dependent and conditioned medium from eosinophils treated with any one of the three cytokines induced increased DNA synthesis. Treatment of fibroblasts with cytokines alone did not induce enhanced DNA synthesis. Eosinophil-conditioned medium also affected fibroblast matrix production. Eosinophil-conditioned medium induced a two-fold increase in soluble and cell-associated fibroblast glycosaminoglycan production and a 76% increase in collagen production. These observations support the concept that eosinophils may be active contributors to the pathophysiology of eosinophil-associated fibrotic disease.Portions of this work were presented at the Society for Investigative Dermatology Annual Meeting, April 1992Supported in part by a grant from the Scleroderma Foundation to S. H. Pincus     

Glycosaminoglycan content in the media of cultured dermal fibroblasts derived from burn scar and normal skin

The glycosaminoglycan (GAG) content of the extracellular matrix of burn scar in humans has been reported to differ from that of normal skin. In order to investigate whether the GAG content altered as a result of functional changes in fibroblasts, the GAG content was determined in culture media of fibroblasts derived from growing burn scar, mature scar, and normal skin tissue. No statistical differences were observed in the population doubling-times of scar and normal skin. Mature scar showed significantly higher values for all the concentrations of uronic acid, hexosamine, and sulfate measured in the glycosaminoglycan, as compared with normal skin values, and the concentrations from growing scar were slightly higher than those for normal skin. The above results may suggest an increase in glycosaminoglycan sulfate synthesis following the hyperplasia of the matrix in burn scar tissue.     

5 alpha-reductase activity in cultured human dermal papilla cells from beard compared with reticular dermal fibroblasts

The activity of 5 alpha-reductase was assessed in cultured human beard dermal papilla cells and reticular dermal fibroblasts to elucidate the mechanism of androgen action in promoting the growth of beards in men. The monolayer was incubated with 50 nM of [1,2-3H]-testosterone. Steroids were extracted from the medium and analyzed by thin layer chromatography. The major metabolite in the beard dermal papilla cells was dihydrotestosterone (DHT), the most potent androgen in the androgen target tissue. By contrast, the amount of DHT formed was similar to that of androstenedione in reticular dermal fibroblasts. The 5 alpha-reductase activity in beard dermal papilla cells was three to five times as high as that in the reticular dermal fibroblasts from the same skin sample. The apparant Michaelis constant of 5 alpha-reductase in the beard dermal papilla cells was 1.0 X 10(-6) M, which was virtually equivalent to that of genital skin fibroblasts, typical androgen target cells. It was 4.0 X 10(-5) M in reticular dermal fibroblasts. By contrast, the activities of 5 alpha-reductase in dermal papilla cells from occipital scalp hair follicles were similar to those of reticular dermal fibroblasts of the same skin samples. These results strongly suggest that the beard dermal papilla cell is an androgen target cell, and that DHT plays a role in the growth of beards in men.     

Phenotypic and functional changes in dermal primary fibroblasts isolated from intrinsically aged human skin

Dermal fibroblasts play a key role in maintaining skin homoeostasis by synthesizing and degrading extracellular matrix components. During ageing, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast ageing was obtained from experiments performed on replicative‐senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age‐related phenotypic and functional alteration of fibroblasts from ‘young’ (<35 years) and ‘old’ (>50 years) donors. Our results brought evidence of the senescent status of ‘old’ fibroblasts by senescence associated β‐galactosidase (SA‐βgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of downregulated genes including cell cycle progression and ECM genes in ‘old’ fibroblasts as well as a subset of upregulated genes involved in senescence features. In ‘old’ fibroblasts, we measured a downregulation of proliferative and contractile capacities of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFβ. Old fibroblasts were also more sensitive to oxidative stress than ‘young’ ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of ‘old’ fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with ageing.     

Proliferation and collagen synthesis by human dermal fibroblasts cultured from different body sites

Distal areas in systemic sclerosis (scleroderma), such as the dorsal skin of the hand are more frequently involved and more indurated than proximal areas. On the contrary, the observation often made after surgical excision or trauma is that distal body areas heal more slowly than proximal areas. A possible explanation may be that dermal fibroblasts from distal body parts are more capable, when stimulated, to synthesize greater or lesser amounts of collagen and proliferate at different rates than dermal fibroblasts from more proximal skin. In this study, cultures of dermal fibroblasts from three different body sites (arm, forearm, and hand) of healthy volunteers were investigated for their proliferative activity and collagen synthesis after stimulation in 3% or 10% fetal bovine serum. No significant differences were observed in cell proliferation or in the relative or absolute collagen synthesis by fibroblasts cultured from the hand, forearm or upper arm. We conclude that other in vivo factors are responsible for the observed differences in fibrosis and healing at different body sites. Moreover, if clonal expansion of different fibroblast phenotypes occurs in these physiologic or disease states, it must be of a magnitude that overcomes the fundamental proliferative and biosynthetic baseline.     

Basic fibroblast growth factor stimulates the proliferation of human dermal fibroblasts via the ERK1/2 and JNK pathways

Background Basic fibroblast growth factor (bFGF, FGF‐2) has been described as a multipotent cytokine that regulates cell growth as well as differentiation, matrix composition, chemotaxis, cell adhesion and migration in numerous cell types. It is known that bFGF stimulates proliferation of cultured fibroblasts. However, the detailed mechanism of fibroblast proliferation induced by bFGF in vitro still remains to be elucidated. Objectives We investigated the precise effects of bFGF on fibroblast proliferation and the signalling pathways responsible for bFGF‐induced proliferation in cultured human dermal fibroblasts (HDFs). Methods HDFs were cultured with bFGF in the presence or absence of specific inhibitors against MAPK signalling pathways including ERK, JNK and p38. The number of cells was counted and immunoblotting findings were examined for the activation of ERK1/2 and JNK. Furthermore, the inhibitory effects of ERK1, ERK2 and JNK1 were proven by the transfection of siRNA. Results bFGF increased the number of HDFs in a dose‐ and time‐dependent manner. The bFGF‐induced proliferation was suppressed by the MEK inhibitors PD98059 and U0126, and the JNK inhibitor SP600125. bFGF increased the phosphorylation levels of ERK1/2 and JNK1. Treatment with ERK1, ERK2 or JNK1 siRNA significantly inhibited bFGF‐induced proliferation. Conclusions This study indicates that ERK1/2 and JNK pathways play an important role in the bFGF‐mediated effect in HDFs. This study also suggests that controlling ERK1/2 and/or JNK signalling may therefore be a new therapeutic approach for the treatment of chronic and untreatable skin ulcers.     

人真皮网状层成纤维细胞在瘢痕疙瘩皮损组织中的表达与分布

目的 探讨人真皮乳头层成纤维细胞(Fp)、网状层成纤维细胞(Fr)和肌成纤维细胞(MFB)在瘢痕疙瘩皮损组织中的表达与分布.方法 2019年5-12月在武汉大学人民医院皮肤科门诊确诊的15例瘢痕疙瘩患者,男8例,女7例,年龄20 ～ 50岁,取皮损组织,以15例年龄匹配的女性乳房整形术正常皮肤组织为对照.采用双重免疫荧...