Expression of endothelial nitric oxide synthase in human eccrine clear cells

Nitric oxide is generated from L-arginine by nitric oxide synthase (NOS), which has at least three isoJorms: endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. Studies by the avidin-biotin immunocomplex method, revealed eNOS immunoreactivity exclusively in the human eccrine clear cells. No eNOS immunoreactivity was observed in the eccrine dark cells or myoepithelial cells. No staining of iNOS or bNOS was observed in the eccrine yland. These findings indicate that NO pUiys a physiological part in the production and/or excretion of sweat in the human skin eccritie gland.     

The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytes

Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression.     

Nitric oxide: a key mediator in cutaneous physiology

Nitric oxide (NO) is a free radical synthesized from l-arginine by a family of NO synthase (NOS) enzymes, all of which are present in the skin, and also by reduction of sweat nitrate. NO synthesis is regulated by NOS activation (eNOS and nNOS) or synthesis (iNOS) and by substrate availability. Elevated arginase concentrations in psoriatic skin suggest that substrate competition may affect NO production. The balance of NO and reactive oxygen species is probably also important in regulating the biological actions of NO. The physiological functions of NO in the skin are being elaborated. NO release is increased following exposure to ultraviolet radiation (UVR); in eNOS null mice, dermal and epidermal apoptosis following UVR exposure is increased. Experiments in which keratinocytes and melanocytes were cocultured show melanogenesis being dependent on keratinocyte-generated NO, and UVR-induced guinea pig pigmentation is delayed following application of a NOS antagonist to the skin. Wound healing is delayed in eNOS and iNOS null mice.     

降钙素基因相关肽对HaCaT细胞一氧化氮合酶表达的影响

目的 研究降钙素基因相关肽对体外培养角质形成细胞HaCaT株神经型一氧化氮合酶(NOS)mRNA、蛋白表达和NO释放的调节作用。方法 应用NO试剂盒(酶法)检测培养细胞上清液中NO水平,RT-PCR和免疫组化(SP)方法检测HaCaT细胞神经型NOSmRNA和蛋白的表达水平。结果 体外正常培养的HaCaT细胞表达和释放基础水平的神经型NOS和NO,降钙素基因相关肽上调HaCaT细胞神经型NOS表达和NO释放,降钙素基因相关肽受体抑制剂CGRP-8-37抑制降钙素基因相关肽的作用。结论 降钙素基因相关肽诱导角质形成细胞表达神经型NOS并促进神经型NO释放。     

Differential expression of nitric oxide synthases in human scalp epidermal and hair follicle pigmentary units: implications for regulation of melanogenesis

BACKGROUND: Nitric oxide (NO) is a ubiquitous gaseous lipophilic molecule generated from the conversion of L-arginine to L-citrulline by the NO synthases (NOSs). Ultraviolet radiation (UVR)-induced NO production appears to stimulate epidermal melanogenesis. However, given their relative protection from UVR, it is unclear whether NO plays a similar role in hair bulb melanocytes. OBJECTIVES: We aimed to identify the expression profiles of the NOS isoforms endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) and of phosphorylated eNOS and nitrotyrosine within the epidermal and follicular melanin units of normal human haired scalp during the hair growth cycle. METHODS: This study employed single and double immunohistochemical and immunofluorescence staining techniques using haired scalp from 10 healthy individuals (six women and four men). RESULTS: Melanocytes in the basal layer of the epidermis expressed eNOS, nNOS and nitrotyrosine. By contrast, melanogenically active melanocytes of the anagen hair bulb were wholly negative for these markers. However, other follicular melanocytes not actively involved in pigment production, including undifferentiated melanocytes located in the outer root sheath and melanocytes surviving the apoptosis-driven hair follicle (HF) regression during catagen/telogen, expressed eNOS, nNOS and nitrotyrosine. While iNOS was only weakly expressed in the basal layer of the human epidermis, it was highly expressed in keratinocytes of the inner root sheath (IRS), where it colocalized with trichohyalin, a differentiation-associated protein of the IRS that requires enzyme-catalysed conversion of arginine to citrulline. CONCLUSIONS: The NOS isoforms and nitrotyrosine are differentially expressed in different cutaneous melanocyte subpopulations. Results of this study suggest a possible role for eNOS, nNOS, iNOS and nitrotyrosine in melanocyte biology, particularly with respect to melanogenesis and melanocyte survival during HF regression. Another example of possible NO involvement in HF biology is the postsynthetic modification of trichohyalin in differentiating keratinocytes of the IRS. These results suggest that NO may influence several aspects of HF biology.     

Distribution of metallothionein in normal and pathological human skin

The expression and distribution of metallothionein (MT) in frozen sections of normal and pathological human skin was studied using the monoclonal antibody L2E3 directed against MT derived from human fetal liver. Immunohistochemical staining of normal fetal and adult skin revealed strong reactivity in basal keratinocytes of epidermis and outer hair root sheath, hair matrix cells and the secretory coil, but not the exocrine portion of eccrine glands; myoepithelial cells around apocrine sweat glands were similarly stained. In epidermal hyperplasia, variable numbers of suprabasal keratinocytes were stained, whereas in interface dermatitis, interrupted staining was found in the basal layer. Weak or scattered staining was observed in squamous tumours, whereas basal cell carcinomas did not show consistent staining. The distribution of MT in normal skin was in line with the germinative role of basal keratinocytes and hair matrix cells, whereas its distribution in hyperplastic epidermis was in line with experimental animal data, and reflected the increase in the germinative pool in these conditions. It is concluded that monoclonal antibody L2E3 may serve as a valuable immunohistochemical marker in diagnostic cutaneous pathology since it labels basal keratinocytes selectively, and since it discriminates between eccrine and apocrine sweat glands.     

Differentiation-associated localization of small prolne-rich rotein in normal and diseased human skin

Summary The expression of SPRR (small proline-rich protein) was investigated in normal human skin and in diseased skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma. Naevus pigmentosus, ichthyosis vulgaris and several inflammatory skin diseases, by immunohistochemical staining. A polyclonal antibody was raised against a synthetic peptide for a C-terminal common region for SPRR l and SPRR 3. In immunoblot analysis, a positive band of 18kDa was detected, which showed the presence of SPRR l in human epidermal keratinocytes. In normal epidermis, positive staining for SPRK was observed in keratinocytes in the granular layer and the uppermost or two spinous cell layers, with no staining of the other spinous or basal layers. The staining was obvious at the cell periphery, weak at the cytoplasm, and absent in the nucleus. Staining was observed in several outer layers of the follicular infundibulum to the isthmus. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, eccrine duct, melanocytes. Langerhans cells or fibroblasts. The arrectores pilorum, striated muscles, muscle layers of vessels, and myoepithelia of eccrine gland, were weakly stained. In psoriatic skin, stained keratinocytes were distributed in the spinous cell layers except for the basal layer, in ichthyosis vulgaris. SPRR was barely expressed in the uppermost living cell layers of the epidermis in epidermolytic hyperkeratosis. degenerated squamous cells widely expressed SPRR. In Darier's disease, dyskeratolic cells were clearly stained. In squamous cell carcinoma, staining was observed in keratotic cells around horny pearls. In basal cell epithelioma, naevus pigmentosus, and malignant melanoma, the tumour cells or naevus cells were not stained. The distribution of SPRR was similar to that of involucrin in normal and several diseased skin, except for ichthyosis vulgaris. We conclude that SPRR is expressed in close association with epidermal differentiation in normal skin and skin diseases. The alteration of the expression of the proteins correlated to terminal differentiation, and differs from disease to disease.     

Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin

Kang-Rotondo CH, Major S, Chiang TM, Myers LK, Kang ES. Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin. Photodermatol Photoimmunol Photomed 1996: 12: 57–65. © Munksgaard, 1996. Ultraviolet B (UVB) irradiation of the skin has been reported to upregulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combined effect of UVB and bradykinin on NOS was therefore examined in a cultured human keratinocyte (KC) line. Activity was determined in KC homogenates by the recovery of [³H]l -citrulline using labeled l -arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterparts were used to determine the isoform(s) in control, UVB, bradykin treated and UVB and bradykinin treated KC. Human KC express NOS activity which is lowest at confluence and highest during proliferation. UVB increased NOS activity when a set dose of irradiation was administered from 32.2–48.3 mJ/cm² but was inhibitory after 64.4 and 80.5 mJ/cm². Thirty min after 10^?6 M bradykinin, NOS activity nearly doubled followed by return of activity to control levels at 60 min. Activity after UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M_r 155 kDa was detected in control cells with the antibody for the constitutive brain enzyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent. When both stimulants were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the expression of NOS activity under unstimulated conditions in human KC in culture is due to the constitutive NOS found in neuronal tissue, bNOS. The recovery of bNOS increased after UVB and after bradykinin while the combination of both resulted in the synergistic increase in bNOS protein with only a marginal further increase in NOS activity.     

Cell surface glycoproteins define distinct states of eccrine gland differentiation

Summary The capability to distinguish eccrine gland cells of the ductal compartment from the secretory portion on the molecular level, as well as from epidermal keratinocytes and other skin cells, is of importance for the study of eccrine differentiation and function. Furthermore, the assessment of differences between the cell systems is useful for the characterization of benign and malignant neoplasms derived from eccrine glands. In the present study, we analysed the expression of four selected epithelial cell surface glycoproteins (gp 80, gp 38, gp 115 and gp 200) in eccrine glands using specific monoclonal antibodies. We found that the glycoproteins are differentially expressed in the ductal compartments and in the secretory portion of the glands, as well as in normal epidermis and other skin cells. This permitted the assignment of specific phenotypes to cells of the ductal compartment and to those of the secretory portion.     

Mammary and extramammary Paget's disease: expression of Ca 15-3, Ka-93, Ca 19-9 and CD44 in Paget cells and adjacent normal skin

The histogenesis of mammary Paget's disease (MPD) and extramammary Paget's disease (EMPD) cells remains controversial. The purpose of this study was to investigate MPD and EMPD immunohistochemically with antibodies to some tumour markers (Ca 15-3, KA-93 and Ca 19-9), and a cell surface receptor for hyaluronate (CD44), as these have been shown to be expressed in normal eccrine or apocrine glands and/or the epidermis, as well as some tumours. Surgically excised, formalin-fixed, paraffin-embedded tissues, or frozen tissues, from seven mammary, five vulvar, two scrolal and two axillary lesions were studied. Paget cells stained strongly with antibodies to Ca 15-3 and KA-93, but did not stain with those to Ca 19-9 and CD44. Staining with the antibody to Ca 15-3 was also observed in the ductal and secretory portions of the eccrine and apocrine glands, and in the sebaceous gland cells. Staining with the antibody to KA-93 was also seen in the apocrine secretory coils, lactiferous duct, epidermal dendritic cells, and cells in the dermal inflammatory infiltrate. Staining with the antibody to Ca 19-9 was observed only in the eccrine duct, and that to CD44 was seen in eccrine secretory cells and epidermal keratinocytes. These findings suggest that the origin of Paget cells may be the secretory cells of apocrine sweat glands (in EMPD) or the luminal lactiferous ducts (in MPD). We found that the antibodies to Ca 15-3 and CD44 were useful in differentiating Paget cells from surrounding keratinocytes, by showing positive and negative immunoreactivity, respectively.     

Identification of novel genes regulated by α-melanocyte-stimulating hormone in murine bone marrow-derived dendritic cells

The vanilloid receptor subtype 1 (VR1/TRPV1) is a non-selective cation channel that binds the vanilloid capsaicin and endogenous cannabinoids. In human skin, VR1 has recently been shown to be expressed by keratinocytes in vitro and in vivo . To determine a precise localization of VR1 in other cutaneous compartments in particular cutaneous nerve fibres, we investigated VR1 immunoreactivity as well as mRNA and protein expression in a series of normal and capsaicin-treated human skin. VR1 immunoreactivity could be observed in cutaneous sensory nerve fibres, mast cells, epidermal keratinocytes, dermal blood vessels, the inner root sheet and infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts and the secretory portion of eccrine sweat glands. Upon RT-PCR and Western blot, the expression of VR1 was confirmed in primary mast cells and keratinocytes from human skin. During capsaicin therapy, VR1-receptor distribution was unchanged, while a reduction of neuropeptides (substance P, calcitonin gene-related peptide) was observed in nerve fibres. After cessation of capsaicin therapy, neuropeptides re-accumulated in skin nerves. In conclusion, VR1 is widely distributed in the skin, suggesting a central role for this receptor, e.g. in nociception and inflammation.     

Protease-Activated Receptor-2 Is Associated with Terminal Differentiation of Epidermis and Eccrine Sweat Glands

Background

Protease-activated receptor 2 (PAR-2) participates in various biological activities, including the regulation of epidermal barrier homeostasis, inflammation, pain perception, and melanosome transfer in the skin.

Objective

To evaluate the basic physiological role of PAR-2 in skin.

Methods

We investigated PAR-2 expression in human epidermis, skin tumors, and cultured epidermal cells using western blot and immunohistochemical analysis. Additionally, we examined the effect of the PAR-2 agonist, SLIGRL-NH2, on cultured keratinocytes.

Results

Strong PAR-2 immunoreactivity was observed in the granular layer of normal human skin and the acrosyringium of the eccrine sweat glands. In contrast, weak PAR-2 immunoreactivity was seen in the granular layer of callused skin and in the duct and gland cells of the eccrine sweat glands. Interestingly, PAR-2 immunoreactivity was very weak or absent in the tumor cells of squamous cell carcinoma (SCC) and syringoma. PAR-2 was detected in primary keratinocytes and SV-40T-transformed human epidermal keratinocytes (SV-HEKs), an immortalized keratinocyte cell line, but not in SCC12 cells. SV-HEKs that were fully differentiated following calcium treatment displayed higher PAR-2 expression than undifferentiated SV-HEKs. Treatment of cultured SV-HEKs with PAR-2 agonist increased loricrin and filaggrin expression, a terminal differentiation marker.

Conclusion

Our data suggest that PAR-2 is associated with terminal differentiation of epidermis and eccrine sweat glands.     

Sulfur mustard downregulates iNOS expression to inhibit wound healing in a human keratinocyte model

BACKGROUND: Increased nitric oxide (NO) synthesized by inducible NO synthase (iNOS) is involved in inflammatory and pathological conditions. iNOS also regulates several biomarkers that accelerate normal wound healing. Effects of exposure to sulfur mustard (SM) on the skin include formation of blisters and slow-healing injuries. Promoting re-epithelialization is a challenging issue in the treatment of the delayed healing of SM-induced skin injuries. OBJECTIVES: To clarify the role(s) of iNOS in wound healing and the effect of SM on iNOS expression in an in vitro wound assay to eventually develop therapies for SM skin injuries. METHODS: A wound was created by scratching normal human epidermal keratinocytes grown in vitro. iNOS expression was monitored by Western blotting, fluorescence microscopy, and real-time RT-PCR. Wound healing was analyzed using digitalized image analysis software. RESULTS: The level of iNOS peaked 24-48h after wounding. SM exposure strongly reduced iNOS protein and mRNA levels. Fluorescence microscopy revealed that induction of iNOS expression by wounding and inhibition of iNOS expression by SM occurred not only in the cells at the wound edge but also in cells in the surrounding area, suggesting that wounding may induce and SM may inhibit release of cytokines that stimulate iNOS expression. iNOS-specific small interfering RNAs caused a marked decrease of iNOS expression irrespective of wounding. Gene silencing also completely inhibited wound healing. CONCLUSION: These results suggest that preventing SM-induced inhibition of iNOS may be a prospective strategy to promote wound healing in SM-exposed skin.     

Merkel cells in human fetal eccrine glands

The presence of human Merkel cells in the eccrine ridges and eccrine germs was studied, using antibodies to simple epithelial keratins, in separated epidermal sheets with attached eccrine ducts. The localization of Merkel cells could be analysed three-dimensionally in the wet, whole-mount of the stained sheets. In the plantar skin of a 12-week-old human fetus, immunoreactive (ir-) Merkel cells were randomly located in the flattened epidermis. In the plantar skin of a 15-week-old human fetus, there was early development of eccrine germs, and Merkel cells were concentrated in eccrine gland ridges. In the plantar skin of a 20-week-old human fetus, eccrine germs were well formed and ir- Merkel cells were located within the developing eccrine ridges and ducts. In the plantar skin of adults, the eccrine concentration of Merkel cells was markedly reduced. Concentration of Merkel cells on the eccrine structures was also observed in the scalp skin of human fetuses. This tendency continued into adult life, although there was a marked reduction in the total number of Merkel cells. These findings suggest that epidermal Merkel cells move down into the eccrine ducts as eccrine germs extend into the mesenchyme. Alternatively, they may develop de novo from the keratinocytes of the eccrine duct. In view of the expression of nerve growth factor receptor in fetal Merkel cells, it is postulated that these eccrine gland Merkel cells play a role in the formation of the periglandular nerve plexus.     

Expression of sarco/endo-plasmic reticulum Ca2+-ATPase type 2 isoforms (SERCA2) in normal human skin and mucosa,and Darier's disease skin

BACKGROUND: The recent report that mutations in ATP2A2, which encodes the Ca2+ transporting sarco/endo-plasmic reticulum pump type 2 isoforms (SERCA2), cause Darier's disease (DD) suggests that SERCA2 plays an important role in epidermal cell adhesion and differentiation. However, no data exist regarding SERCA2 expression in normal human skin, mucosa and DD. OBJECTIVES: We have therefore investigated SERCA2 expression in normal human skin (40 samples), oral and vaginal mucosa (13 samples) and DD lesional skin (six samples). MATERIALS AND METHODS: These investigations were performed with a mouse monoclonal antibody specific for human SERCA2, using a standard ABC immunoperoxidase technique. RESULTS: SERCA2 was expressed in all specimens. SERCA2 expression was pronounced in the subnuclear aspect of basal epidermal keratinocytes, with variable suprabasal expression. SERCA2 expression was also observed in the infundibulum and outer root sheath of hair follicles; germinative and mature cells of sebaceous glands; secretory coil and duct of eccrine glands; apocrine gland cells, and arrector pili muscle. Fibroblasts and blood vessels (endothelium and muscle) expressed SERCA2, whereas nerves did not. SERCA2 expression was observed throughout oral and vaginal mucosa. In DD skin, strong SERCA2 positivity was detected in the basal, suprabasal and acantholytic lesional cells. Perilesional DD skin was comparable to normal skin. CONCLUSIONS: These findings support the hypothesis that SERCA2 is an important player in cutaneous biology, and provide baseline data that will facilitate the design and interpretation of functional studies of cutaneous SERCA2.     

Expression and localization of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in human epidermal appendages: a comparison study by immunofluorescence

Background.  Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles in neovascularization and development of tissues. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We have previously shown that keratinocytes from human normal skin express VEGFRs. This poses the question of whether these receptors are also expressed by epidermal appendages, as epidermal appendages are lined with epithelial cells.
Objective.  To investigate the expression of VEGFR-2 compare with VEGF in epidermal appendages, including hair follicles, eccrine sweat glands and sebaceous glands.
Methods.  Monoclonal antibodies to VEGF and VEGFR-2 were used for immunohistochemical examination of cryostat-cut sections of normal human skin specimens from 11 donors undergoing cosmetic surgery.
Results.  Immunoreactivities for VEGF and VEGFR-2 principally showed parallel intense expression in anagen hair follicle (including outer root sheat, inner root sheath, dermal papillae epidermal matrix), sebaceous glands (ductal and secretory portions) and eccrine sweat glands (ductal and secretory portions), respectively. In particular, abundant expression of VEGF was found in the follicular basement membrane zone surrounding the bulb matrix and in the ductal and secretory portions of eccrine sweat glands.
Conclusion.  A potential VEGF/VEGFR-2 autocrine pathway may be defined by the coexpression of VEGF and VEGFR-2 in human skin epidermal appendages.     

中波紫外线对人角质形成细胞iNOS的作用

目的观察中波紫外线(UVB)照射对体外培养正常人角质形成细胞(HKC)中诱导型一氧化氮合成酶(iNOS)的作用,探讨紫外线引起皮肤细胞急性损伤的发病机制。方法体外培养HKC,用免疫荧光染色和蛋白印迹技术检测照射后不同时间细胞内iNOS的水平。结果HKC经UVB照射后,免疫荧光法检测到细胞膜胞浆侧有iNOS,蛋白印迹法检测到细胞提取总蛋白中分子量为130kDa的蛋白条带可以被抗iNOS多克隆抗体所识别,二者结果相符合。结论UVB照射可诱导体外培养的HKC在胞浆内产生iNOS,iNOS蛋白的表达可能与紫外线引起的皮肤急性炎症反应有关。