A clinicopathological analysis of forkhead box A1 (FOXA1) and estrogen receptor alpha expression in extramammary Paget's disease.

Objectives: Extramammary Paget's disease (EMPD) is a neoplastic skin disease of unknown etiology. EMPD is frequently associated with forkhead box A1 (FOXA1) expression, which correlates with the expression of estrogen receptor alpha (ER). FOXA1 regulates the transcriptional activity of ER and may function cooperatively in the tumorigenesis of breast cancer. Methods: We performed immunohistochemical staining for FOXA1 and ER using tissue samples from 16 patients with EMPD. Results: The nuclei of Paget cells isolated from each of the 16 patients with EMPD (100%) were strongly FOXA1-positive, and the FOXA1 staining intensity was similar across all samples. ER staining was detected in the nuclei of Paget cells originating from seven patients with EMPD (44%), and the ER staining intensity varied between these patients. Conclusions: In the present study, we confirmed that EMPD was frequently associated with FOXA1 expression. However, ER expression varied between patients and did not always coincide with FOXA1 expression. No clear relationship was observed between ER expression, the intensity of ER staining, or EMPD metastasis and prognosis. However, the results indicate that hormone-dependent cancer therapy may be effective in patients with ER-positive EMPD.

Melanin accumulation in dermal stem cells deteriorates their exosome-mediated skin basement membrane construction in solar lentigo.

Solar lentigo (SL) is a hyperpigmented macule that occurs in sun-exposed areas and is characterized by the accumulation of melanin pigment in the epidermis. On the contrary, melanin-incorporated macrophages have also been identified in the dermis, which is thought to be caused by melanin transfer due to disruption of the basement membrane, but the detailed mechanism remains unclear. In this study, we analysed SL lesions by pathological methods and examined the mechanism of melanin accumulation in the dermis using cultured skin models in vitro. First, we observed a significant decrease in type IV collagen (COL4), a major component of the basement membrane, in SL lesions. The basement membrane is known to be formed by the interaction of keratinocytes and dermal cells. Therefore, we constructed skin models containing fibroblasts or dermal stem cells and examined their effects on basement membrane formation. The results showed a markedly enhanced production of COL4 mediated by dermal stem cell-derived exosomes. The analysis of melanin localization in the SL dermis revealed that CD163-positive macrophages and CD271-positive dermal stem cells both took up melanin pigment. Exosomes of dermal stem cells incorporating melanosomes were less effective in promoting COL4 expression. These findings suggest that while the promotion of COL4 production in keratinocytes by dermal stem cell-derived exosomes is important for maintaining basement membrane homeostasis, this mechanism is disrupted in SL lesions, leading to chronic melanin accumulation in the dermis.

Enhanced Type I Collagen Synthesis in Fibroblasts by Dermal Stem/Progenitor Cell-Derived Exosomes.

The self-duplication and differentiation of dermal stem cells are essential for the maintenance of dermal homeostasis. Fibroblasts are derived from dermal stem cells and produce components of connective tissue, such as collagen, which maintains the structure of the dermis. Cell-cell communication is required for the maintenance of tissue homeostasis, and the role of exosomes in this process has recently been attracting increasing attention. Dermal stem cells and fibroblasts have been suggested to communicate with each other in the dermis; however, the underlying mechanisms remain unclear. In the present study, we investigated communication between dermal stem/progenitor cells (DSPCs) and fibroblasts via exosomes. We collected exosomes from DSPCs and added them to a culture of fibroblasts. With the exosomes, COL1A1 mRNA expression was up-regulated and dependent on the Akt phosphorylation. Exosomes collected from fibroblasts did not show the significant up-regulation of COL1A1 mRNA expression. We then performed a proteomic analysis and detected 74 proteins specific to DSPC-derived exosomes, including ANP32B related to Akt phosphorylation. We added exosomes in which ANP32B was knocked down to a fibroblast culture and observed neither Akt phosphorylation nor enhanced type I collagen synthesis. Additionally, an immunohistochemical analysis of skin tissues revealed that ANP32B expression levels in CD271-positive dermal stem cells were lower in old subjects than in young subjects. These results suggest that DSPCs promote type I collagen synthesis in fibroblasts by secreting exosomes containing ANP32B, which may contribute to the maintenance of skin homeostasis; however, this function of DSPCs may decrease with aging.

Age-related decrease in responsiveness of CD271-positive skin stem cells to growth factors.

Previous studies have demonstrated that the numbers of interfollicular epidermal stem cells (IFE-SCs) and dermal stem cells (DSCs) decrease with age and that this decrease is attributed to the age-related deterioration of skin homeostatic functions and the delay in wound healing. Meanwhile, functional decline in the stem cells is also considered to be responsible for the deteriorated skin homeostatic functions and the delayed wound healing associated with ageing. In the present study, we focused on epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) signalling and fibroblast growth factor-2/fibroblast growth factor receptor (FGF2/FGFR) signalling to analyse the age-related changes. Immunohistological analysis revealed that the expressions of EGFR and FGFR1 declined in IFE-SCs and DSCs with age, respectively. Additionally, IFE-SCs and DSCs isolated from the skin samples of elderly subjects exhibited lowered responsiveness to EGF and FGF2, respectively. These results suggest that the lowered responsiveness of the skin stem cells to growth factors may be a factor involved in the age-related deterioration of skin regenerative functions during wound healing and skin homeostatic functions. We hope that homeostatic and wound healing functions in the skin could be maintained if the decreased expressions of EGFR and FGFR1 in IFE-SCs and DSCs, respectively, can be suppressed.

Increase in inhibin beta A/Activin-A expression in the human epidermis and the suppression of epidermal stem/progenitor cell proliferation with aging.

BACKGROUND: Age-related thinning and reduced cell proliferation in the human epidermis are associated with the accumulation of senescent cells and decreases in the number and function of epidermal stem cells. OBJECTIVE: This study examined the expression of INHBA/Activin-A in human epidermis and expression differences with age, and the effect of Activin-A on epidermal stem/progenitor cells. METHODS: Immunohistochemical staining was used to analyze age-related changes in the expression of INHBA/Activin-A in the epidermal tissue of young and old subjects. Epidermal INHBA/Activin-A expression levels, epidermal morphology, and the number of epidermal stem/progenitor cells or proliferating cells were investigated using older abdominal skin samples. The effects of Activin-A on the development of a three-dimensional (3D) reconstructed epidermis and cell proliferation were also assessed. RESULTS: INHBA/Activin-A expression levels in the human epidermis increased with age, although they varied among individuals. In the epidermis of older abdominal skin samples, INHBA/Activin-A expression levels negatively correlated with epidermal thickness, the rete ridge depth and the interdigitation index. The proportion of epidermal stem/progenitor cells and proliferating cells decreased with increases in INHBA/Activin-A expression levels. Activin-A had no effect on the differentiation of keratinocytes in the 3D-reconstructed epidermis; however, thinning of the 3D epidermis was noted. Moreover, the addition of Activin-A inhibited the proliferation of epidermal stem/progenitor cells in a concentration-dependent manner. CONCLUSIONS: Age-related increased in INHBA/Activin-A expression levels were observed in the human epidermis, and may contribute to epidermal thinning and decreases in the number of epidermal stem/progenitor cells and proliferative activity.

UVA causes dysfunction of ETBR and BMPR2 in vascular endothelial cells, resulting in structural abnormalities of the skin capillaries.

BACKGROUND: Capillary structural abnormalities cause skin disorders. Mottled redness, i.e., skin redness unevenness, may appear on the sun-exposed skin, suggesting capillary structural abnormalities, although its mechanism remains unclear. OBJECTIVE: To observe the capillary structures in the sun-exposed skin where skin redness unevenness is likely to occur, and clarify the mechanism of capillary structural abnormalities. METHODS: The tissue structures in the skin with skin redness unevenness were observed by LC-OCT. Subsequently, immunostaining of the sun-exposed skin where skin redness unevenness is often observed, was performed. Vascular endothelial cells were UV-irradiated to analyze the expression and functions of genes involved in the capillary structures and morphogenesis. RESULTS: The skin with skin redness unevenness exhibited scattering of dilated tubular tissue and disturbance of distribution uniformity. Immunostaining of the sun-exposed skin that were more likely to be exposed to UV rays also revealed similarly disorder of capillary structures. In addition, UVA-irradiated vascular endothelial cells exhibited increased expression of ETBR, involved in telangiectasia, decreased expression of BMPR2, involved in the morphogenesis and maintenance of the blood vessels, and reduced migration of the capillaries. CONCLUSION: UV rays alter ETBR and BMPR2 expression in the skin capillaries, and cause partial dilation and decreased migration, resulting in capillary structural abnormalities and causing skin redness unevenness.

Clinical Usefulness of a Modified Mohs' Technique and Topical Application of Zinc Oxide Powder for Treating Skin Infiltration Caused by Unresectable Malignant Tumors.

Background: Infiltrative lesions of the skin caused by unresectable malignant tumors reduce the quality of life of patients significantly due to the presence of bleeding, exudate, pain, and/or malodor. Objective: We compared the efficacy of a modified Mohs' technique and topical application of a starch powder containing zinc oxide as palliative treatments for skin lesions caused by unresectable tumors in our hospital. Design: This is a retrospective study. Settings/Subjects: This study included nine patients who were treated for skin-infiltrating lesions caused by unresectable malignant tumors at our hospital in Japan from April 2008 to December 2019. Measurements: Mohs' paste or zinc oxide powder (50%) was applied to the infiltrative skin lesions. Arterial embolization was performed before the application of the Mohs' paste for patients at risk for arterial hemorrhage. Patients were evaluated for pain, tumor size, bleeding, wound exudate, and malodor. Results: Both treatments were useful for alleviating symptoms, such as tumor size, local bleeding, malodor, and exudate in patients with unresectable malignant tumors. Pain was reduced in patients treated with Mohs' paste for 1 hour as compared with those treated for 24 hours. Conclusions: Effective management of skin infiltrative lesions can be achieved by using a modified Mohs' technique, topical application of starch powder containing zinc oxide, and arterial embolization to reduce the vascularization of the tumors.

Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin.

INTRODUCTION: The skin is comprised of various kinds of cells and has three layers, the epidermis, dermis and subcutaneous adipose tissue. Stem cells in each tissue duplicate themselves and differentiate to supply new cells that function in the tissue, and thereby maintain the tissue homeostasis. In contrast, senescent cells accumulate with age and secrete senescence-associated secretory phenotype (SASP) factors that impair surrounding cells and tissues, which lowers the capacity to maintain homeostasis in each tissue. Previously, we found Gremlin 2 (GREM2) as a novel SASP factor in the skin and reported that GREM2 suppressed the differentiation of adipose-derived stromal/stem cells. In the present study, we investigated the effects of GREM2 on stem cells in the epidermis and dermis. METHODS: To examine whether GREM2 expression and the differentiation levels in the epidermis and dermis are correlated, the expressions of GREM2, stem cell markers, an epidermal differentiation marker Keratin 10 (KRT10) and a dermal differentiation marker type 3 procollagen were examined in the skin samples (n = 14) randomly chosen from the elderly where GREM2 expression level is high and the individual differences of its expression are prominent. Next, to test whether GREM2 affects the differentiation of skin stem cells, cells from two established lines (an epidermal and a dermal stem/progenitor cell model) were cultured and induced to differentiate, and recombinant GREM2 protein was added. RESULTS: In the human skin, the expression levels of GREM2 varied among individuals both in the epidermis and dermis. The expression level of GREM2 was not correlated with the number of stem cells, but negatively correlated with those of both an epidermal and a dermal differentiation markers. The expression levels of epidermal differentiation markers were significantly suppressed by the addition of GREM2 in the three-dimensional (3D) epidermis generated with an epidermal stem/progenitor cell model. In addition, by differentiation induction, the expressions of dermal differentiation markers were induced in cells from a dermal stem/progenitor cell model, and the addition of GREM2 significantly suppressed the expressions of the dermal differentiation markers. CONCLUSIONS: GREM2 expression level did not affect the numbers of stem cells in the epidermis and dermis but affects the differentiation and maturation levels of the tissues, and GREM2 suppressed the differentiation of stem/progenitor cells in vitro. These findings suggest that GREM2 may contribute to the age-related reduction in the capacity to maintain skin homeostasis by suppressing the differentiation of epidermal and dermal stem/progenitor cells.

Senescent cell removal via JAG1-NOTCH1 signalling in the epidermis.

Emerging evidence has pointed to the noxious effects of senescent cells in various tissues, and senescent cells in the epidermis are known to accumulate with age. We hypothesized that there is a mechanism by which senescent cells in the epidermis are preferentially removed and that the function of such removal mechanism declines as age increases. In this study, we investigated whether Notch signalling is involved in such senescent cell removal. We found that Notch1 receptor was expressed more highly in p16INK4a-positive senescent cells than in surrounding cells in human epidermis both in young and old subjects. On the other hand, the expression of its ligand JAG1 was decreased in the epidermis of aged subjects. When normal epidermal cells and UVB-irradiated senescent cells were mixed and three-dimensional reconstructed epidermis was developed in vitro, the senescent cells were preferentially removed from the basal layer and located in the upper layer. We also found that the depletion of senescent cells from the basal layer was suppressed by JAG1 knockdown in normal cells or using a Notch signalling inhibitor. From these results, Notch signalling may be involved in senescent cell removal in the epidermis and the age-related decrease of JAG1 expression in the basal layer may lead to accumulation of senescent cells owing to reduced activation of Notch signalling.

Efficacy and safety of treatment with anti-tumor necrosis factor-α drugs for severe impetigo herpetiformis.

Impetigo herpetiformis (IH) is a rare pustular dermatosis. It can be life-threatening for both the mother and fetus and often causes therapeutic problems. However, there is no specific guideline for the treatment of IH and the evidence regarding the efficacy of treatments for IH has not been established. Herein, we report two cases of IH, which were successfully treated with anti-tumor necrosis factor (TNF)-α drugs. The serum levels of the drugs in the infants and mothers were examined using enzyme-linked immunosorbent assay (ELISA). Case 1 was a 35-year-old, gravida 2, para 1, female patient in week 20 of pregnancy; she was treated with adalimumab (ADA) until delivery. Case 2 was a 26-year-old, gravida 1, para 0, female patient in week 30 of pregnancy; she was treated with certolizumab pegol (CZP) until delivery. In both cases, the skin lesions started regressing considerably after administration of the biologic agents. We examined the serum levels of the biologic agents in the mothers and infants using ELISA. In case 1, the ADA serum level in the infant was as high as that in the mother at birth; it then decreased below the lower limit of quantification at week 12 post-delivery. In case 2, the CZP serum level in the infant was below the lower limit of quantification at birth. In this report, we revealed that biologic agents could be an effective treatment for severe IH and that CZP treatment can be considered safe for the mothers and fetuses.

SASP-induced macrophage dysfunction may contribute to accelerated senescent fibroblast accumulation in the dermis.

Recently, increasing attention has been paid to senescence-associated secretory phenotype (SASP), a phenomenon that senescent cells secrete molecules such as inflammatory cytokines and matrix metalloproteinases (MMPs), due to its noxious effects on the surrounding tissue. Senescent cells in the blood and liver are known to be properly depleted by macrophages. In the dermis, accumulation of senescent cells has been reported and is thought to be involved with skin ageing. In this study, to elucidate the clearance mechanism of senescent cells in the dermis, we focused on macrophage functions. Our co-culture experiments of senescent fibroblasts and macrophages revealed a two-step clearance mechanism: first, TNF-α secreted from macrophages induces apoptosis in senescent fibroblasts, and then, dead cells are phagocytosed by macrophages. Furthermore, it was suggested that SASP factors suppress both of the two steps of the senescent cell clearance by macrophages. From these findings, normally senescent cells in the dermis are thought to be removed by macrophages, but when senescent cells are excessively accumulated owing to oxidative stress, ultraviolet (UV) ray or other reasons, SASP was suggested to suppress the macrophage-dependent clearance functions and thereby cause further accumulation of senescent cells.

Increase of gremlin 2 with age in human adipose-derived stromal/stem cells and its inhibitory effect on adipogenesis.

INTRODUCTION: Adipose-derived stromal/stem cells (ASCs) have attracted attention as a promising material for regenerative medicine. Previously, we reported an age-related decrease in the adipogenic potential of ASCs from human subjects and found that the individual difference in this potential increased with age, although the mechanisms remain unclear. Recently, other groups demonstrated that a secreted antagonist of bone morphogenetic protein (BMP) signaling, Gremlin 2 (GREM2), inhibits the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts and the adipogenesis of 3T3-L1 cell. Here, we examined the effects of GREM2 on the differentiation of ASCs into adipocytes. METHODS: To examine changes in GREM2 expression levels with age, immunohistochemistry was performed on subcutaneous adipose tissues from subjects 12-97 years of age. Next, GREM2 gene expression levels in ASCs collected from subjects 5-90 years of age were examined by RT-PCR, and the change with age and correlation between the expression level and the adipogenic potential of ASCs were analyzed. In addition, to assess whether GREM2 affects adipogenesis, ASCs (purchased from a vendor) were cultured to induce adipogenesis with recombinant GREM2 protein, and siRNA-induced GREM2 knockdown experiment was also performed using aged ASCs. RESULTS: In adipose tissues, GREM2 expression was observed in cells, including ASCs, but not in mature adipocytes, and the expression level per cell increased with age. GREM2 expression levels in ASCs cultured in vitro also increased with age, and the individual differences in the level increased with age. Of note, partial correlation analysis controlled for age revealed that the adipogenic potential of ASCs and the GREM2 gene expression level were negatively correlated. Furthermore, based on a GREM2 addition experiment, GREM2 has inhibitory effects on the adipogenesis of ASCs through activation of Wnt/ß-catenin signaling. On the other hand, GREM2 knockdown in aged ASCs promoted adipogenesis. CONCLUSIONS: The GREM2 expression level was confirmed to play a role in the age-related decrease in adipogenic potential observed in ASCs isolated from adipose tissues as well as in the enhancement of the individual difference, which increased with age. GREM2 in adipose tissues increased with age, which suggested that GREM2 functions as an inhibitory factor of adipogenesis in ASCs.

UV irradiation-induced DNA hypomethylation around WNT1 gene: Implications for solar lentigines.

Wnt/ß-catenin signalling promotes melanogenesis in melanocytes and also induces melanocytogenesis from melanocyte stem cells (McSCs). Previous study reported that WNT1, a ligand which activates Wnt/ß-catenin signalling pathway, was more highly expressed in the epidermis at SLs than in normal skin areas, suggesting that WNT1 causes hyperpigmentation. To elucidate the mechanism by which WNT1 expression is increased in SLs, we examined the methylation of 5-carbon of cytosine (5mC), that is 5-methylcytosine (5mC) level, in a region within the WNT1 promoter; the methylation of the region was known to negatively regulate WNT1 gene expression. We used an immortalized cell line of human interfollicular epidermal stem cells to analyse the effect of UVB irradiation on DNA methylation level of WNT1 promoter and found that UVB irradiation caused demethylation of WNT1 promoter and promoted WNT1 mRNA expression. It was also found that UVB irradiation reduced the expression of DNA methyltransferase 1 (DNMT1), an enzyme responsible for maintaining methylation patterns during cell division. Pathological analysis of SLs and non-SL regions in the human skin revealed that both DNMT1 expression and 5mC level were decreased at SLs compared to non-SL skins. Furthermore, bisulphite sequencing showed that the methylated CpG level in WNT1 promoter was also lower at SLs than in non-SL skins. Thus, in the skin exposed to a high amount of UV rays, excessive expression of WNT1 is thought to be caused by the demethylation of WNT1 promoter, and the upregulated WNT1 promotes melanocytogenesis and melanogenesis, then resulting in SL formation.

CXCL12 regulates differentiation of human immature melanocyte precursors as well as their migration.

Melanocyte stem cells (McSCs) are localized in the bulge region of hair follicles and supply melanocytes, which determine hair color by synthesizing melanin. Ectopic differentiation of McSCs, which are usually undifferentiated in the bulge region, causes depletion of McSCs and results in hair graying. Therefore, to prevent hair graying, it is essential to maintain McSCs in the bulge region, but the mechanism of McSC maintenance remains unclear. To address this issue, we investigated the role of CXCL12, a chemokine which was previously suggested to induce migration of melanocyte lineage cells, as a niche component of McSCs. Immunohistological analysis revealed that CXCL12 was highly expressed in the bulge region of human hair follicles. CXCL12 mRNA expression level was significantly lower in white hairs plucked from human scalps than in black hairs. CXCL12 attracted the migration of early-passage normal human epidermal melanocytes (eNHEMs), an in vitro model of McSCs, which had characteristics of immature melanocyte precursors. We also found that CXCL12 suppressed their differentiation. These results suggest that CXCL12 regulates differentiation of McSCs as well as their proper localization, and maintaining McSCs by regulating CXCL12 expression level in the bulge region may be a key to preventing hair graying.

Extracellular proteoglycan decorin maintains human hair follicle stem cells.

Hair follicle stem cells (HFSC) are localized in the bulge region of the hair follicle and play a role in producing hair. Recently, it has been shown that the number of HFSC decreases with age, which is thought to be a cause of senile alopecia. Therefore, maintaining HFSC may be key for the prevention of age-related hair loss, but the regulatory mechanisms of HFSC and the effects of aging on them are largely unknown. In general, stem cells are known to require regulatory factors in the pericellular microenvironment, termed the stem cell niche, to maintain their cell function. In this study, we focused on the extracellular matrix proteoglycan decorin (DCN) as a candidate factor for maintaining the human HFSC niche. Gene expression analysis showed that DCN was highly expressed in the bulge region. We observed decreases in DCN expression as well as the number of KRT15-positive HFSC with age. In vitro experiments with human plucked hair-derived HFSC revealed that HFSC lost their undifferentiated state with increasing passages, and prior to this change a decrease in DCN expression was observed. Furthermore, knockdown of DCN promoted HFSC differentiation. In contrast, when HFSC were cultured on DCN-coated plates, they showed an even more undifferentiated state. From these results, as a novel mechanism for maintaining HFSC, it was suggested that DCN functions as a stem cell niche component, and that the deficit of HFSC maintenance caused by a reduction in DCN expression could be a cause of age-related hair loss.

Laminin-332 regulates differentiation of human interfollicular epidermal stem cells.

Interfollicular epidermal stem cells (IFE-SCs) have self-renewal and differentiation potentials, and maintain epidermal homeostasis. Stem cells in vivo are regulated by the surrounding environment called niche to function properly, however, IFE-SC niche components are not fully understood. In order to elucidate the mechanisms of keeping epidermal homeostasis and of skin aging, and also to develop new therapeutic technologies for skin diseases, we searched for niche factors that regulate IFE-SCs. We found that laminin-332, a basement membrane component, was highly expressed at the tips of the dermal papillae, where IFE-SCs are localized, and that the stem cells by themselves expressed laminin-332. Knockdown of laminin-332 during the culture of IFE-SC-model cells to construct 3-dimensional epidermis in vitro resulted in failure to form proper structure, although no significant change was observed in either cell growth or apoptosis. Pre-coating of the culture insert with laminin-332 restored the normal formation of 3-dimensional epidermis. From these results, it was shown that laminin-332 is an essential niche component for the proper differentiation of IFE-SCs.