Ultrastructural observation of possible nerve endings in rat sebaceous gland

Summary Neural elements within the parenchyma of the sebaceous gland have not been reported previously. Nerve endings have been observed only in the connective tissue surrounding the gland or in close association with the undifferentiated basal cells.In this study, electron microscopy revealed the possible presence of nerve endings (or terminal portions of neural elements) in the suprabasal level of functional sebaceous glands of pinnae of white rats. Morphologically, there are two distinct types of nerve endings. Type 1 is bordered by a membrane of relatively irregular contour and contains a single mitochondrion, various-sized vesicles, numerous microtubules, fine neurofilament-like fibrils, and occasional ribosome-like granules. Type II is also bordered by a membrane, but its contour was relatively smooth and rounded. Moreover, Type II contains many mitochondria, varying in size, density, and the arrangement of cristae. While ribosome-like granules are scattered throughout the structure in relative abundance, there are scarcely any fine neurofilament-like fibrils or microtubules. Whether these two structures are sensory or autonomic fibers could not be determined by electron microscopic examination.     

Immunohistochemical markers of human sebaceous gland differentiation

Cryostat sections of human skin were stained with monoclonal antibodies to involucrin, a range of cytokeratins, epithelial membrane antigen (EMA), and an ovarian cystadenocarcinoma antibody (OM1) to identify combinations of antibodies that could be used to discriminate between basal and differentiated sebocytes and other cell types present in the pilosebaceous unit. Both the EMA and OM1 monoclonal antibodies specifically recognized differentiated sebocytes. No staining of basal sebocytes or other epidermal cell types was seen. Differentiated (but not basal) sebocytes were also stained by a cytokeratin 10 antibody (LH2). Conversely, the basal sebocytes were recognized by an antibody specific to basal keratinocytes (LH6). Cells of the sebaceous duct stained with both LH2 and LH6 and also with the anti-involucrin monoclonal antibody. Cytokeratin 4 has been detected in sebaceous glands by protein analysis but has not previously been detectable immunohistochemically. We show by immunofluorescence after limited proteolysis that cytokeratin 4 epitopes are distributed in all sebaceous gland cells, including the duct cells.     

Embigin is a fibronectin receptor that affects sebaceous gland differentiation and metabolism

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Development and homeostasis of the sebaceous gland

The important role of epidermal appendages especially the sebaceous gland has only recently been recognized. In particular, it has been convincingly shown that normal development and maintenance of the sebaceous gland are required for skin homeostasis since atrophic sebaceous glands and disturbances in sebaceous lipid composition result in major defects of the physiological barrier and maintenance of the skin. Consequently, it is important to unravel the signaling network controlling proper sebaceous lineage differentiation in mammalian skin and to understand the underlying mechanisms leading to severe skin diseases, including abnormal proliferation and differentiation of the gland, defects of the lipid metabolism and barrier, as well as sebaceous tumor formation. Over the last years, results from transgenic and knock out mouse models manipulating distinct signaling pathways in the skin as well as the detailed analysis of human sebaceous gland-derived cell lines provided new insights into crucial mediators balancing proliferation and differentiation of the sebaceous gland. Here, we discuss our current knowledge of in vivo mechanisms of sebaceous gland development, maintenance and disorders and highlight recent contributions to the field of sebaceous gland biology.     

Endogenous retinoids in the hair follicle and sebaceous gland

Vitamin A and its derivatives (retinoids) are critically important in the development and maintenance of multiple epithelial tissues, including skin, hair, and sebaceous glands, as shown by the detrimental effects of either vitamin A deficiency or toxicity. Thus, precise levels of retinoic acid (RA, active metabolite) are needed. These precise levels of RA are achieved by regulating several steps in the conversion of dietary vitamin A (retinol) to RA and RA catabolism. This review discusses the localization of RA synthesis to specific sites within the hair follicle and sebaceous gland, including their stem cells, during both homeostasis and disease states. It also discusses what is known about the specific roles of RA within the hair follicle and sebaceous gland. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

Protective action of resveratrol in human skin: possible involvement of specific receptor binding sites

Background

Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006) J Pharmacol Exp Ther 318:238–245). The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol.

Methods and Findings

Using human skin tissue, we report here the presence of specific [³H]-resveratrol binding sites (K_D  = 180 nM) that are mainly located in the epidermis. Exposure of HaCaT cells to the nitric oxide free radical donor sodium nitroprusside (SNP; 0.3–3 mM) resulted in cell death which was reduced by resveratrol (EC₅₀  = 14.7 µM), and to a much lesser extent by the resveratrol analogue piceatannol (EC₅₀  = 95 µM) and epigallocatechin gallate (EC₅₀  = 200 µM), a green-tea derived polyphenol. The protective action of resveratrol likely relates to its anti-apoptotic effect since at the same range of concentration it was able to reduce both the number of apoptotic cells as well as mitochondrial apoptotic events triggered by SNP.

Conclusion

Taken together, these findings suggest that resveratrol, by acting on specific polyphenol binding sites in epidermis, may be useful to prevent skin disorders associated with aging.     

A lectin-binding immunoassay indicates a possible glycosylated growth hormone in the human pituitary gland

By means of a lectin binding immunoassay we detected a glycosylated human growth hormone-immunoreactive substance in the human pituitary gland. The assay involves reacting the test substance with concanavalin A immobilized on a solid support followed by treatment with human growth hormone antibodies. Quantitation is achieved by reaction of the concanavalin A-glycoprotein-antibody complex with an 125I-labeled second antibody. Specificity of the assay for a glycosylated human growth hormone was indicated by inhibition of binding by N-acetyl-D-glucosamine. Furthermore, use of antiserum that had been absorbed with human growth hormone reduced the amount of radioactivity bound to the tubes. Western blotting of pituitary immunoprecipitates after gel electrophoresis in sodium dodecyl sulfate revealed human growth hormone-immunoreactive components that also reacted with concanavalin A.     

Comparative studies of the ultrastructure of the sebaceous gland

Three-dimensional reconstructions and ultrastructural evidence on the sebaceous glands of man and domestic animals indicate that sebum is produced from a column of developing and degenerating cell populations derived from peripheral progenitor cells at the base of the lobe in a manner analogous to hair growth. The remainder of the peripheral cells apparently have no direct involvement with sebocyte production; those towards the neck of the gland contribute keratin to the secretory product.     

Characterization of specific high-affinity receptor for human lymphotoxin

The specific cell surface receptors for lymphotoxin (LT) which are expressed on murine fibroblast L.P3 cells, a subline of L929 cells, were found to consist of a single class of specific high-affinity receptors with a dissociation constant (Kd) of 3.8 X 10(-10) M and a density of 5.8 X 10(3) sites/cell. Similarly, murine fibroblast L929 cells, human melanoma A375 cells and human cervical carcinoma HeLa-S3 cells had about 7.2 X 10(3), 3.5 X 10(3), and 6.6 X 10(3) sites/cell with Kd values of 1.4 X 10(-10), 0.5 X 10(-10), and 1.1 X 10(-10) M, respectively. Among the LT receptor-positive cell lines, there was no direct correlation between the level of specific LT binding and the sensitivity to the cytotoxic or cytostatic effect of LT. Cross-linking of 125I-LT to the cell surface receptors with disuccinimidyl suberate, followed by two-dimensional gel electrophoresis of the cell lysate, revealed two kinds of LT-LT receptor complexes with molecular weights of 70 and 97 kDa, and having the same pI value of 6.8. Cell-bound 125I-LT was internalized within 1 h and degraded intracellularly, and finally secreted into the medium within a few hours. Appropriate concentrations of LT and interferon gamma (IFN gamma) showed synergistic cytotoxicity toward murine fibroblast L.P3 cells and human monocytoma U937 cells, but these cytokines were only slightly cytotoxic individually. Preincubation of these cells with IFN gamma increased the total number of LT receptors without any significant change in the dissociation constant or in the molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia

Long-term survival still eludes most patients with leukemia and non-Hodgkin's lymphoma. No approved therapies target the hallmark of the B cell, its mIgM, also known as the B-cell receptor (BCR). Aptamers are small oligonucleotides that can specifically bind to a wide range of target molecules and offer some advantages over antibodies as therapeutic agents. Here, we report the rational engineering of aptamer TD05 into multimeric forms reactive with the BCR that may be useful in biomedical applications. Systematic truncation of TD05 coupled with modification with locked nucleic acids (LNA) increased conformational stability and nuclease resistance. Trimeric and tetrameric versions with optimized polyethyleneglycol (PEG) linker lengths exhibited high avidity at physiological temperatures both in vitro and in vivo. Competition and protease studies showed that the multimeric, optimized aptamer bound to membrane-associated human mIgM, but not with soluble IgM in plasma, allowing the possibility of targeting leukemias and lymphomas in vivo. The B-cell specificity of the multivalent aptamer was confirmed on lymphoma cell lines and fresh clinical leukemia samples. The chemically engineered aptamers, with significantly improved kinetic and biochemical features, unique specificity and desirable pharmacological properties, may be useful in biomedical applications.     

The mineralocorticoid receptor may compensate for the loss of the glucocorticoid receptor at specific stages of mammary gland development

To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and beta-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce beta-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.     

Testosterone-dependent primer pheromone production in the sebaceous gland of male goat

To test the hypothesis that the primer pheromone responsible for inducing the "male effect" is produced in the sebaceous gland androgen dependently, we examined the correlation between morphological changes of sebaceous glands and the pheromone activity in skin samples taken from castrated goats that had been treated with testosterone. Five castrated goats were implanted s.c. with testosterone capsules to maintain physiological levels of plasma testosterone for four weeks. Skin samples were obtained from the head region on Day 0 (the day of testosterone implant), Day 7, Day 14, Day 28 (the day of testosterone removal), Day 36, Day 42, and Day 56. Matched blood samples were also collected for measurement of testosterone concentration. The pheromone activity of the ether-extracts of the upper dermal layer containing sebaceous glands was assessed by its stimulatory effect on the hypothalamic GnRH pulse generator, which was monitored for changes of specific multiple unit activity (MUA) in ovariectomized estradiol-primed goats as described previously. The sebaceous gland enlarged during the testosterone treatment but reduced in size after testosterone removal. The pheromone activity first appeared in 2 out of 5 goats on Day 7 and in all the 5 goats by Day 28. Fourteen days after testosterone removal (Day 42), the pheromone activity was no longer detectable in any of the 5 goats. In short, the sebaceous gland size and the pheromone activity shifted almost in parallel. The present results provide strong support for the view that the primer pheromone is produced testosterone dependently in the sebaceous gland of the male goat.     

Stem cell dynamics in sebaceous gland morphogenesis in mouse skin

The hair follicle (HF) and the sebaceous gland (SG) constitute the two integral parts of the pilosebaceous unit and significantly contribute to the barrier function of mammalian skin. Considerable progress has been made in our understanding how HF formation is regulated. However, the development of the SG is poorly understood, both at the molecular and cellular level. Here, we investigate the process of SG morphogenesis and the dynamics of its cellular organisation in more detail. The spatial and temporal organisation of distinct stem and progenitor compartments was analysed during morphogenesis of the pilosebaceous unit in mouse tail epidermis. Our experiments reveal a dynamic expression pattern for diverse HF stem cell marker molecules including Sox9 and Lrig1. Surprisingly, Sox9 and Lrig1 are initially coexpressed by epidermal progenitor cells and are confined to different regions within the pilosebaceous unit when the specification of the sebocyte cell lineage takes place. We demonstrate that SG development at the distal part of the HF is driven by asymmetric cell fate decision of Lrig1 positive stem cells, whereas MTS24/Plet1 positive precursor cells seem not to play a role in this process. Importantly, our data clearly show that distinct stem and progenitor compartments are established at different time points of development. By studying the process of SG morphogenesis more precisely, we discovered that the two prominent SGs attached to one tail HF originate from one small cluster of sebocyte cells. Finally, we show regional specificity for HF patterning and spatio-temporal control of the underlying molecular signals initiating the development of the pilosebaceous unit.     

A possible role for dephosphorylation in glucocorticoid receptor transformation

Addition of bovine intestinal alkaline phosphatase to mouse AtT-20 cell cytosol increases the rate of glucocorticoid receptor transformation, as evidenced by a change in sedimentation rate from 9.1S to 5.2S. Acid phosphatases are completely ineffective in this regard. Alkaline phosphatase-promoted receptor transformation is both time- and dose-dependent. A variety of phosphatase inhibitors are effective in inhibiting this process, the most potent being transition metal oxyanions such as molybdate, tungstate, and arsenate. The ability of the various inhibitors to suppress alkaline phosphatase-promoted receptor transformation does not correspond well with their potencies for inhibiting para-nitrophenyl phosphate hydrolysis. However, a better correspondence between the inhibition of endogenous receptor transformation and total cytosolic phosphatase activity is observed, and both sodium fluoride and glucose-1-phosphate inhibit endogenous receptor transformation. The protease inhibitors phenyl-methylsulfonyl fluoride and antipain have no effect on receptor transformation. Surprisingly, leupeptin is effective in inhibiting alkaline phosphatase-promoted receptor transformation. Although this raises the possibility of a contaminating protease activity in the alkaline phosphatase enzyme preparation, treatment of covalently affinity-labeled receptor with the enzyme shows no proteolysis of the receptor or any other non-specifically labeled cytosolic protein. Thus, it is possible that a novel action of leupeptin, unrelated to its protease-inhibitory activity, may be involved in the suppression of receptor transformation. The studies presented here suggest that dephosphorylation of some component in cytosol is involved in the destabilization of receptor subunit interactions, resulting in glucocorticoid receptor transformation.