Transforming growth factor beta and connective tissue growth factor are involved in the evolution of nevus of Nanta

We report a 59-year-old Japanese male with nevus of Nanta on the face. Histological examination revealed a nest of nevus cells in the dermis and ossification beneath the nevus. Positive staining for transforming growth factor-beta and connective tissue growth factor suggested the involvement of these growth factors in the ossification of nevus of Nanta.     

Keratinocytes inhibit expression of connective tissue growth factor in fibroblasts in vitro by an interleukin-1alpha-dependent mechanism

The wound healing process concludes with downregulation of fibroblast activity. Clinical observations suggest that the regenerating epidermis suppresses this activity. An important regulator of fibroblast activity is the fibrogenic cytokine connective tissue growth factor. We hypothesized that epidermal keratinocytes may affect fibroblast activity via this cytokine. We demonstrate keratinocyte-mediated suppression of connective tissue growth factor at both the mRNA and protein levels by around 50% or more when fibroblasts were cultured in multiwell plates with keratinocyte cultures in accompanying semipermeable cell culture inserts, or stimulated by keratinocyte-conditioned media. Both basal and transforming-growth-factor-beta1-stimulated levels of connective tissue growth factor were inhibited. A 3 h coculture period with keratinocytes was sufficient to suppress connective tissue growth factor expression by fibroblasts, but the inhibition developed over a time period of around 16 h. The putative keratinocyte-derived factor(s) responsible for these effects was found to be soluble and stable. By analyzing cytokines secreted by keratinocytes we identified interleukin-1alpha as a potent inhibitor of connective tissue growth factor mRNA expression in fibroblasts. Involvement of this cytokine in keratinocyte-mediated connective tissue growth factor suppression was confirmed by using anti-interleukin-1alpha antibodies. Tumor necrosis factor alpha or prostaglandins did not appear to be involved. In conclusion, our results indicate that interleukin-1alpha secretion by keratinocytes provides a mechanism for the downregulation of connective tissue activity during the end-stage of wound healing, when epithelia coverage has developed over the wound area.     

The role of chronic inflammation in cutaneous fibrosis: fibroblast growth factor receptor deficiency in keratinocytes as an example

Fibrosis is associated with a variety of skin diseases and causes severe aesthetic and functional impairments. Functional studies in rodents, together with clinical observations, strongly suggest a crucial role of chronic injury and inflammation in the pathogenesis of fibrotic diseases. The phenotype of mice lacking fibroblast growth factor (FGF) receptors 1 and 2 in keratinocytes supports this concept. In these mice, a defect in keratinocytes alone initiated an inflammatory response, which in turn caused keratinocyte hyperproliferation and dermal fibrosis. As the mechanism underlying this phenotype, we identified a loss of FGF-induced expression of claudins and occludin, which caused abnormalities in tight junctions with concomitant deficits in epidermal barrier function. This resulted in severe transepidermal water loss and skin dryness. In turn, activation of keratinocytes and epidermal γδ T cells occurred, which produced IL-1 family member 8 and S100A8 and S100A9. These cytokines attracted immune cells and activated fibroblasts, resulting in a double paracrine loop through production of keratinocyte mitogens by dermal cells. In addition, a profibrotic response was induced in fibroblasts. Our results highlight the importance of an intact epidermal barrier for the prevention of inflammation and fibrosis and the role of chronic inflammation in the pathogenesis of fibrotic diseases.     

Treatment of generalized scleroderma with inhibitors of connective tissue formation.

103 patients suffering from generalized scleroderma were studied in order to assess the effect of treatment with inhibitors of connective tissue formation. 93 patients with generalized scleroderma were given D-penicillamine, benzyl-penicillin-diethylamino-ethyl-ester hydroiodide, adrenal glucocorticoids, dextro-thyroxine, hydralazine, and "mixed treatment" (one or several of the drugs in consecutive courses, or concurrently). The effect of dextrothyroxine could not be evaluated in this study. No improvement could be seen after adrenocortical steroid therapy. Hydralazine seemed to be effective. D-penicillamine improved 25 of 34 treated patients; penicillin hydroiodide 12 out of 16. The dermal sclerosis of 6 patients regressed completely; in 16, sclerosis regressed with the exception of finger sclerosis; in 32, partial regression was registered; 20 had their progression arrested, but there was no regression; in 19 cases, there was no effect whatsoever. The prognosis seemed to be better for young than for old people. The age at onset was lower in the better groups. The higher the total dose, the better the results. The length of the treatment course is probably of some significance. The short-lasting cases had better prospects than the longer lasting. Ten untreated patients of this material and 11 patients seen earlier showed continued progression. Side effects leading to discontinuation of the drugs were seen in a substantial number of patients, especially after D-penicillamine. Twelve deaths could not be related to the treatments.     

The role of ERK and JNK signaling in connective tissue growth factor induced extracellular matrix protein production and scar formation

CCN2 plays an important role in the pathogenesis of hypertrophic scars (HTSs). Although CCN2 is involved in many fibroproliferative events, the CCN2 induction signaling pathway in HTSs is yet to be elucidated. Here, we first investigated the effect of the mitogen-activated protein kinases (MAPKs) on CCN2-induced α-smooth muscle actin (α-SMA) and collagen I expression in human HTS fibroblasts (HTSFs). Then, we established HTSs in a rabbit ear model and determined the effect of MAPKs on the pathogenesis of HTSs. MAPK pathways were activated by CCN2 in HTSFs. Extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors significantly inhibited CCN2-induced expression of α-SMA and collagen I in HTSFs. In the rabbit ear model of the HTS, JNK and ERK inhibitors significantly improved the architecture of the rabbit ear scar and reduced scar formation on the rabbit ear. Our results indicate that ERK and JNK mediate collagen I expression and scarring of the rabbit ear, and may be considered for specific drug therapy targets for HTSs.     

Differential expression of connective tissue growth factor and extracellular matrix proteins in lichen sclerosus

Background The histopathology of lichen sclerosus (LS) suggests abnormalities in extracellular matrix (ECM) composition. Objectives We aimed to investigate the expression pattern of ECM proteins and related growths factors and Smad signal transducers in LS as compared with healthy skin. Methods To assess the expression of decorin, biglycan, versican, perlecan, fibronectin, dermatopontin, extracellular matrix protein 1 (ECM‐1), matrix metalloproteinase 1, tissue inhibitor of metalloproteinase 1, connective tissue growth factor (CTGF), transforming growth factor β1, and Smad‐3 protein, real‐time RT‐PCR and immunohistochemistry were performed on skin specimens obtained from the genital region of healthy subjects (n = 10) as well as LS patients (n = 26). Results Median mRNA as well as mean protein expression of biglycan, versican, fibronectin, and ECM‐1 was significantly higher in LS when compared with healthy controls. Both mRNA and protein CTGF expression observed in LS was significantly higher than in controls. CTGF mRNA expression significantly correlated with mRNA expression of biglycan, versican and fibronectin. Conclusions Expression of ECM proteins (e.g. proteoglycans, ECM‐1) and CTGF is altered in LS. TGF‐ß/Smad‐3 independent up‐regulation of CTGF may induce accumulation of ECM proteins and maintain fibrosis in chronic LS.     

Induction of connective tissue growth factor expression by sphingosylphosphorylcholine in cultured human skin fibroblasts

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In an effort to find downstream effectors of SPC, we performed microarray analysis and found that the expression of the gene for connective tissue growth factor (CTGF) was significantly affected in human skin fibroblasts cultured in vitro. Northern blot analysis showed that SPC markedly induced CTGF mRNA expression in a dose- and time-dependent manner. Consistent with this result, Western blot analysis also showed that SPC significantly induced the CTGF production. Pretreatment with cycloheximide did not prevent the CTGF induction by SPC, indicating that SPC stimulates CTGF mRNA expression without the increased synthesis of a regulatory protein. Inhibition by pretreatment with Y27632, but not by PD98059 (a mitogen-activated protein kinase 1/2 inhibitor) and LY294002 (a phosphatidylinositol 3-kinase inhibitor), indicated that rho-kinase pathway was involved in SPC-induced CTGF expression. Together, these results reveal the potential importance of CTGF induction as a downstream event in SPC-induced cellular responses.     

Differential expression of connective tissue growth factor gene in cutaneous fibrohistiocytic and vascular tumors

Connective tissue growth factor (CTGF) is a member of a family of immediate early gene products that may play an important role during tissue regeneration, wound repair and skin fibrosis. In this study, CTGF gene expression in mesenchymal tumors was investigated by in situ hybridization and CD34 antigen expression was studied by means of immunohistochemical staining. CTGF mRNA was expressed in fibroblasts of all nine dermatofibromas examined, but five of seven dermatofibrosarcoma protuberans (DFSP) or two cases of malignant fibrous histiocytoma were negative for its expression. In contrast, CD34 antigen was expressed only in DFSP. In vascular tumors, CTGF mRNA was expressed in pyogenic granuloma but not in angiosarcoma. In addition, the endothelial cells in angiolipoma and angioleiomyoma, but not in venous lake, expressed CTGF mRNA. These vascular lesions were all positive for CD34 expression. Tumors of other origins were negative for CTGF mRNA. Our findings indicated that benign fibroblasts and/or vascular endothelial cells have the capability to express CTGF mRNA when activated, but these cells lose this ability when they achieve malignant potency. Thus, examination of CTGF gene expression may be useful for differentiating between benign and malignant mesenchymal tumors, or to determine the origin of the tumors in connective tissue.     

Possible role of connective tissue in epidermal neoplasia

Interactions between epidermal cells have been defined within a proposed mathematical model of mammalian skin. Testing the model in a computer suggests that in highly proliferating conditions of the epidermis competition for cell space in the basal layer may be sufficient to generate considerable forces in the papillary dermis. Data shown from human and pig epidermal hyperplasia indicate that basal cells are submitted to considerable lateral forces and that these and not dermal hyperplasia are the forces responsible for the increasingly folded dermo-epidermal junction. When the model was examined in condition of persistently high mitolic rate it was found that it could remain stable only if new connective tissue synthesis was not induced by the developing papillary tension. This complex and counterproductive relationship that may occur between epidermis and dermis and its possible role in the development of neoplasia are discussed.     

Immunofluorescence studies in progressive systemic sclerosis (scleroderma) and mixed connective tissue disease

Immunofluorescence (IF) investigations of the skin were performed in thirty patients with progressive systemic sclerosis (scleroderma) and eight patients with mixed connective tissue disease (MCTD). The results show that speckled epidermal nuclear immunoglobulin deposition occurs not only in MCTD but also in true scleroderma. Granular IgM deposition at the dermo-epidermal junction of light-exposed skin was detected in both groups of patients, but six of eight MCTD patients also showed a granular IgM band in non-exposed skin. Antinuclear antibodies (ANA) were demonstrated in the sera of 96% and 100% of patients with scleroderma and MCTD respectively. The pattern of nuclear IF staining in scleroderma included dense fine speckles, large coarse speckles, threads, nucleolar and centromere staining. In MCTD, by contrast, the ANA staining pattern consisted of threads. The significance of ANA titres and immunological specificities for the in vivo reaction of serum ANA with epidermal nuclear antigens is discussed.     

Pseudoscleroderma associated with lung cancer: correlation of collagen type I and connective tissue growth factor gene expression

Pseudoscleroderma as a paraneoplastic syndrome is a rare disease. We report here a patient with lung cancer (undifferentiated squamous cell carcinoma), who developed acrosclerosis. Using in situ hybridization, marked expression of alpha1(I)-collagen and connective tissue growth factor (CTGF) mRNA was found in fibroblasts scattered throughout the dermis. However, transforming growth factor (TGF)-beta1 expression was not detected. The pattern of CTGF gene expression and collagen synthesis was similar to that in systemic scleroderma. The absence of TGF-beta1 mRNA could indicate that tumour-derived factors induce the expression of CTGF.     

Basic fibroblast growth factor treatment for skin ulcerations in scleroderma

We report the use of topical application of recombinant human basic fibroblast growth factor (rhbFGF) to successfully treat therapy-resistant, chronic leg ulcers in scleroderma. Endothelial cell FGF receptors are directly stimulated by bFGF; also, bFGF promotes the regeneration of capillary-rich granulation tissue. We conclude that topical bFGF may be a powerful new pharmacologic tool for treating severe skin ulcers.     

Reduced response of scleroderma fibroblasts to fibroblast growth factor

Summary Reactivity of scleroderma fibroblasts to lymphoid cell-derived fibroblast growth factor (FGF) was assessed in this study. The fibroblasts from the sclerotic lesion failed to respond to FGF, whereas those from scleroedematous lesions responded equally to normal fibroblast. Response of the fibroblast from sclerotic lesion was also lower than that of the fibroblast from mature scar. Fibroblasts obtained from three different layers of healthy skin, papillary dermis, reticular dermis, and reticular-subcutaneous layer, responded equally to FGF, whereas the fibroblast of reticular dermis from sclerotic lesion failed to respond to FGF. It is suggested that the fibroblast of reticuar dermis in scleroderma is variously activated by some unknown factors, so that they do not have enough reserve to respond to further stimuli.     

积雪苷体外对瘢痕疙瘩成纤维细胞增殖及结缔组织生长因子表达的影响

目的 探讨积雪苷体外对瘢痕疙瘩成纤维细胞增殖和对结缔组织生长因子（CTGF）表达的影响。方法 取手术切除的瘢痕疙瘩组织作成纤维细胞的原代培养,加入不同浓度积雪苷,观察细胞的形态变化,MTT法检测细胞活性,免疫组化和Western印迹法检测积雪苷对瘢痕疙瘩成纤维细胞结缔组织生长因子表达的影响。结果 细胞形态学观察显示,经不同浓度积雪苷处理的成纤维细胞呈现明显的抑制及凋亡征象。积雪苷在1 ～ 100 mg/L范围内,在24、48、72 h时积雪苷浓度与细胞活性抑制率均呈正相关,r分别为0.95、0.90和0.92,P值均 ＜ 0.01;且各浓度不同时间段细胞活性抑制率间单因素方差分析,P值均 ＜ 0.01。瘢痕疙瘩成纤维细胞中CTGF呈强阳性表达,而经积雪苷处理瘢痕疙瘩成纤维细胞48 h后,CTGF表达有所减弱,每100个成纤维细胞中CTGF表达阳性细胞数均值未加药组为73个,1 mg/L积雪苷组为54个,10 mg/L积雪苷组为46个,未加药组与1 mg/L、10 mg/L积雪苷组比较,差异均有统计学意义（t值分别为4.34和6.26,P值均 ＜ 0.01）;1 mg/L积雪苷组与10 mg/L积雪苷组比较,差异亦有统计学意义（t = 1.95,P ＜ 0.05）。Western印迹显示,积雪苷作用48 h后,成纤维细胞中CTGF的表达量比未加药的细胞明显减弱,且表达量随药物剂量的加大有递减趋势。结论 积雪苷能有效抑制瘢痕疙瘩成纤维细胞的增殖和结缔组织生长因子的表达。     

The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines

Please cite this paper as: The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines. Experimental Dermatology 2010; 19 : 865–872. Abstract: Solar lentigines (SLs) are hyperpigmentary lesions presented on sun‐exposed areas of the skin and associated with ageing. The molecular mechanism of SL initiation is not completely understood. Ultraviolet B (UVB) stimulates keratinocytes to produce interlukin‐1 alpha (IL‐1α), which then induces keratinocyte growth factor (KGF) secretion; therefore, we examined their possible roles in the induction of SLs. We found that KGF increases pigment production in both pigmented epidermal equivalents and human skin explants. In addition, UVB exposure increases KGF expression, and KGF treatment induces tyrosinase (TYR) expression in primary melanocytes. The KGF‐induced pigmentary changes were confirmed using pigmented Yucatan swine, and human skins grafted onto immuno‐deficient mice. In both model systems, the topical treatment with KGF, alone or in combination with IL‐1α, resulted in the in vivo formation of hyperpigmentary lesions with increased pigment deposition and elongated rete ridges, which resemble the histological features of human SLs. Preliminary immunohistochemical analysis of human skins showed a moderate increase in KGF, and a strong induction in KGF receptor (KGFR) in SL lesions. In summary, KGF increases pigment production and deposition in vitro and in vivo. Moreover, we show for the first time the in vivo generation of hyperpigmentary lesions with histological resemblance to human SLs and indicate the involvement of KGF/KGFR in the molecular pathology of human SLs.     

Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. [14C]Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of [14C]proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.