ARTICLE |
Correspondence to: Joseph E. Mazurkiewicz, Center for Neuropharmacology and Neuroscience, Mail Code A-136, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. E-mail: jmazurki@.amc.edu
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Summary |
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In murine skin, after depilation-induced anagen, there was a differential spatial and temporal expression of pro-opiomelanocortin (POMC) mRNA, of the POMC-derived peptides ß-endorphin, ACTH, ß-MSH, and -MSH, and of the prohormone convertases PC1 and PC2 in epidermal and hair follicle keratinocytes and in the cells of sebaceous units. Using a combination of in situ hybridization histochemistry and immunohistochemistry, we found cell-specific variations in the expression of POMC mRNA that were consistent with immunoreactivities for POMC-derived peptides. Cells that contained POMC peptide immunoreactivity (IR) also expressed POMC mRNA, and where the IR increased there was a parallel increase in mRNA. The levels of PC1-IR and PC2-IR also showed cell-specific variations and were present in the same cells that contained the POMC peptides. Based on the cleavage specificities of these convertases and on the spatial and temporal expression of the convertases and of ACTH, ß-endorphin, ß-MSH, and
-MSH, we can infer that the activities of PC1 and PC2 are responsible for the cell-specific differential processing of POMC in murine skin. (J Histochem Cytochem 48:905914, 2000)
Key Words:
skin, pro-opiomelanocortin, POMC, prohormone convertases, hair cycle, ACTH, -MSH, ß-MSH, ß-endorphin
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Introduction |
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Pro-opiomelanocortin (POMC) is a 30-kD precursor protein that is posttranslationally processed to yield a number of neuropeptides, including ACTH, the melanocyte-stimulating hormones (-MSH, ß-MSH,
-MSHs), ß-lipotropins, and endorphin opioids (ß-endorphin,
-endorphin,
-endorphin) (
In the pituitary and hypothalamus, posttranslational proteolysis of POMC is performed by the prohormone convertases PC1 and PC2. Cleavage by PC1 results primarily in the production of ACTH and ß-lipotropin and, to a minor extent, ß-endorphin, whereas cleavage by PC2 elicits ß-endorphin and N-terminally extended corticotropin containing the joining peptide and either -MSH or desacetylated-
-MSH. Removal of the terminal dipeptide from ß-endorphin-[131] to yield ß-endorphin-[127] is mediated solely by PC2 (
To directly study the relationship between POMC gene transcription and the production of POMC peptides after depilation-induced anagen in murine skin, we used in situ hybridization (ISH) histochemistry to identify sites of POMC mRNA expression and immunohistochemistry (IHC) to identify the cells that contained POMC peptides (ß-endorphin, ACTH, ß-MSH, and -MSH), and the prohormone convertases PC1 and PC2.
All of the analyses in the present study were performed on tissue sections that were taken from the same skin samples at each time point, thus permitting direct correlations of expression and processing of the POMC precursor. The results reported here can also be directly correlated with an earlier biochemical and molecular biology study in which we had demonstrated by RT-PCR that POMC mRNA is expressed constitutively in murine skin (
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Materials and Methods |
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Tissue Preparation
All animal experiments were performed in compliance with guidelines of the National Institutes of Health for the use of laboratory animals. C57 BL/6 mice (female, syngenic, 68 weeks old) were purchased from Charles River (Kingston, NY) housed with 12-hr light/darkness periods, and fed with water and mouse chow ad libitum. Anagen was induced in anesthetized mice (30 mg/kg sodium pentobarbital) by hair depilation using a warm beeswax/rosin mixture as described previously (
Immunohistochemistry
Sections were deparaffinized with xylene and rehydrated through a decreasing ethanol series to phosphate-buffered saline (PBS; 150 mM NaCl/10 mM sodium phosphate, pH 7.3). Endogenous peroxidase activity was blocked by incubation with 1% H2O2 in absolute methanol for 5 min. Nonspecific protein binding was blocked with a solution of 5% nonfat dry milk, 0.1% Triton X-100 in PBS (Blotto) for 5 min. Samples were then washed in PBS three times for 5 min each.
Immunoperoxidase Detection
POMC antigens were detected by the indirect avidinbiotinperoxidase method (ABC standard; Vector Laboratories, Burlingame, CA). Color was developed with 3, 3' diaminobenzidine. Primary antisera were diluted in blocking buffer, applied to tissue sections that were encircled with a PAP pen (Research Products International; Mount Prospect, IL), and incubated for 90 min at room temperature (RT). Rabbit anti-ACTH (1:600) and rabbit anti-ß-endorphin (1:500) were gifts from Dr. R.G. Allen (Oregon Health Sciences University); rabbit anti ß-MSH (1:500) was a gift of Dr. S.H. Pomerantz (University of Maryland). Optimal dilutions and incubation times had been determined in a previous study (-MSH (20%) and with ACTH (5%) (
MSH (1:600) was from Chemicon International (Temecula, CA). According to the manufacturer, there is no crossreactivity of this antiserum with other POMC peptides. Controls (not shown) consisted of tissue incubated with nonimmune sera or with primary antibody that had been incubated with 0.1 mmol/liter concentrations of ß-endorphin, ACTH (Peninsula Laboratories; Belmont, CA), ß-MSH (gift of Dr. G. Davila, Yale University), or a mixture of porcine pituitary POMC peptides (gift of Dr. J. Pawelek, Yale University). In all cases at the dilutions used, the immunoreactivty was abolished when the primary antiserum was absorbed with its respective peptide. If significant amounts of unprocessed POMC precursor had been present in the tissue sections, blocking with the specific peptide alone would not have eliminated crossreactivity. Some sections were counterstained with Gill's hematoxylin. Photographs were taken on an Olympus BX40 upright microscope interfaced with a SONY Mavigraph video printer. Photographs were digitized and the figures were assembled and labeled in Photoshop.
Immunofluorescence Detection
The prohormone convertases PC1 and PC2 were detected by indirect immunofluorescence and confocal laser scanning microscopy (CLSM). Rabbit anti-PC1-Ab888 and anti-PC2-Ab1159 (1:100) were gifts from Dr. B.A. Eipper (Johns Hopkins University School of Medicine). The secondary antibody was a goat anti-mouse IgAlexa488 (Molecular Probes; Eugene, OR). Specimens were analyzed on a NORAN-OZ CLSM interfaced with a Nikon Diaphot 200 inverted microscope equipped with a PlanApo x60, 1.4 NA oil-immersion objective lens. The specimens were first examined by conventional widefield fluorescence microscopy to assess the specificity of the immunostaining procedure and to identify samples with the highest fluorescence intensity. The instrument settings on the CLSM for brightness, contrast, laser power, and slit size were optimized for the brightest sample to ensure that the CLSM was set for its full dynamic range and that no images were saturated. These settings were used for all subsequent image collection for a particular immunostaining experiment. This permits relative quantitation of the cellular content of these antigens for comparative purposes, and any differences can be readily discerned directly from the images.
In Situ Hybridization Histochemical Localization
Skin sections from telogen, and at 3, 5, and 10 days post anagen induction were subjected to ISH. POMC mRNA was localized in skin and pituitary sections according to procedures outlined in
Tissue sections (5 µm) mounted on Superfrost/Plus microscope slides (Fisher Scientific; Pittsburgh, PA) were deparaffinized in xylene and rehydrated in an ethanol series to DEPC (diethypyrocarbonate)-treated H2O. Sections were transferred to 0.1 M Tris-HCl, 50 mM EDTA, pH 8.0, prewarmed to 37C. Proteinase K from a stock solution in the same buffer was added to a final concentration of 0.001% and incubated for 30 min at 37C. Sections were then washed briefly (~1 min) in DEPC-treated H2O, then in freshly prepared 0.1 M triethanolamine buffer, pH 8.0, and acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min. Slides were then rinsed in 2 x SSC (sodium citrate saline). Hybridization was carried out overnight with sense or antisense cRNA probe (0.3 ng/µl/kb of sequence length). Thirty µl of hybridization solution was put on each section and overlaid with a paraffin coverslip that just covered the section. Slides were placed in a plastic box containing absorbent paper saturated with a solution of 50% formamide in 2 x SSC, the box sealed to prevent evaporation, and the slides incubated overnight at 42C. After hybridization, the slides were transferred to 2 x SSC to remove the coverslips and were washed for 5 min. Slides were transferred to STE (0.5 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for 1 min, then incubated in STE containing RNase A (40 µg/ml) for 30 min at 37C. The sections were then taken through stringency washes as follows: 2 x SSC, 50% formamide at 50C for 5 min; 1 x SSC at RT for 5 min; 0.5% SSC at RT for 5 min. The specificity of the probe was confirmed by substitution of DIG-labeled sense RNA or by pretreatment of tissue sections with RNase (50 µg/ml for 30 min at 37C) before application of DIG-labeled antisense RNA. Mouse pituitary gland tissue prepared in the same way as the skin was used as a positive ISH control.
Immunologic Detection of DIG-labeled Probes
Hybridized DIG-labeled probes were demonstrated either with anti-DIG-labeled alkaline phosphatase (DIG-AP) or with an indirect immunogold system.
DIG-AP Detection of Probes.
After washing in TBS (150 mM NaCl in 100 mM Tris-HCl, pH 7.5) for 1 min and incubation in BoehringerMannheim blocking solution for 30 min, sections were incubated with a mouse monoclonal anti-digoxigenin antibody (BoehringerMannheim) diluted 1:500 in TBS for 1 hr at RT in a humidified chamber. Sections were washed in TBS for 5 min, then in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2 for 10 min. Substrate solution for color detection consisted of 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2, 1 mM levamisole, plus 5 µl NBT/ml and 3.5 µl X-Phosphate/ml. Sections were incubated overnight under 500 µl of substrate solution in the dark at 4C. The reaction was stopped by placing slides in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. Slides were coverslip-mounted with glycerol gelatin (
Indirect Immunogold Detection of DIG-labeled Probes. Sections were first blocked in Blotto for 5 min and then incubated overnight at 4C with sheep anti-DIG antibody (BoehringerMannheim) diluted 1:50 in PBS, 2% nonfat dry milk. Sections were washed three times for 5 min in PBS, incubated with biotinylated rabbit anti-sheep Ig diluted 1:200 for 1 hr at RT, followed by three washes in PBS of 5 min each. Sections were then incubated with 10-nm gold-conjugated goat anti-biotin antibody (EMS Sciences; Worthington, PA) diluted 1:200 in PBS, 1% BSA, 0.01% Tween-20, 0.01% Triton X-100 for 1 hr at RT. Sections were washed in PBS three times for 5 min each and incubated in 1% glutaraldehyde in PBS for 20 min at RT. Sections were then rinsed in PBS once for 5 min followed by washing in dH2O, five times for 1 min each, to remove salts. Sections were then subjected to immunogoldsilver staining (IGSS) to amplify the immunogold reaction according to the method described in
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Results |
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POMC mRNA Is Differentially Expressed by Separate Skin Cells at Different Times After Anagen Induction
We had shown by RT-PCR that POMC mRNA is expressed constitutively in murine skin and is upregulated during anagen (
ISH histochemistry for POMC mRNA on murine pituitary using an alkaline phosphatase-based immunodetection method of DIG-labeled cRNAs resulted in intense staining in the intermediate lobe, light staining in the anterior lobe, and negative staining of the posterior lobe. This was the expected pattern of staining for the positive control (Fig 1). With this procedure, POMC message was readily detected in the skin at Day 5 after induction of anagen and thereafter. However, no staining was detected with this technique at telogen or Day 1, and only weak staining was detected at Day 3 (data not shown). We therefore used a more sensitive IGSS method to detect the anticipated lower levels of mRNA in these cells while still retaining the advantage of high spatial resolution. The reaction product of the IGSS procedure is deposited as a very fine precipitate in the cytoplasm of the cells, closely resembling the silver grains in an autoradiograph. The density of the precipitate can be related directly to the amount of mRNA present in a cell.
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At telogen, POMC mRNA levels were below the level of detection by ISH despite the use of silver enhancement. However, by Day 1 after induction, POMC mRNA was readily detectable in sebaceous units and in scattered keratinocytes in the epidermis. The staining in sebaceous units was very intense at Day 3 and remained so for the rest of the hair growth cycle (Fig 2A and Fig 2C). The number of POMC mRNA-positive epidermal keratinocytes continued to increase throughout anagen, with all epidermal keratinocytes containing signal by Day 10 after induction. The increase in hybridization signal occurred in aggregates of cells in patches in the epidermis, until all were positive (Fig 2A and Fig 2C). Message for POMC was detectable by Day 3 after anagen induction in keratinocytes in the hair follicle, in the region immediately adjacent to the sebaceous gland. Hybridization signal was very sparse in both inner and outer root sheath cells in the follicle below the sebaceous unit at Day 5 (Fig 2B) and increased steadily in cells of both sheaths as the cycle progressed (illustrated for Day 10 in Fig 2D). POMC mRNA was not detected in cells of the hair follicle matrix (Fig 3).
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POMC Peptides Displayed a Differential Cellular Localization and Temporal Expression During the Hair Cycle
For IHC, we used antibodies to ACTH, ß-endorphin, ß-MSH, and -MSH to demonstrate that the entire POMC precursor is translated. All four of these POMC peptides were present in murine skin. Immunolocalization of ACTH, ß-endorphin, and ß-MSH is illustrated in Fig 4.
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IHC for the POMC products ACTH, ß-endorphin, ß-MSH, and -MSH showed a differential cellular distribution and temporal expression during induction of the hair cycle (Table 1). ACTH immunoreactivity was present in epidermal and follicle keratinocytes. Immunoreactivity was absent at telogen, with immunopositivity appearing at anagen Day 3 and increasing to Day 15. In the later part of the cycle, epidermal keratinocytes showed a decrease in ACTH-IR but follicular cell-IR remained constant. In addition to the obvious cellular immunoreactivity for ACTH, there was strong immunoreactivity at the epidermaldermal junction (Fig 4B and Fig 4C) This staining was apparent at Day 3 and increased in intensity throughout the hair cycle. None of the other peptides displayed the latter immunoreactive pattern.
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ß-MSH immunoreactivity was present in epidermal and follicle keratinocytes and in sebocytes. ß-MSH-IR was absent at telogen. By Day 3, epidermal keratinocytes, some sebocytes, and sebaceous gland duct cells were immunopositive. By Day 5, outer root sheath cells were also positive. The intensity of staining increased dramatically through Day 10 after induction and returned to telogen levels by Day 15.
In contrast to ACTH and ß-MSH, ß-endorphin staining was limited to sebocytes. It was present at telogen and increased significantly in staining intensity throughout anagen.
At telogen, -MSH immunoreactivity was absent in keratinocytes and was weakly present in sebocytes. Immunoreactivity was present at Day 3 in epidermal and follicle keratinocytes and increased through Day 6, after which it remained strong through Day 15. Staining of sebocytes did not change and remained at telogen level throughout the hair cycle.
PC1 and PC2 Displayed a Differential Cellular Localization and Temporal Expression During the Hair Cycle
As with POMC mRNA and POMC-derived peptides, PC1 and PC2 also displayed a differential temporal and spatial pattern of expression. PC1 and PC2 were present at very low levels in epidermal keratinocytes in telogen; PC1-IR was greater than PC2-IR (Fig 5). Immunoreactivity for both convertases was increased in anagen VI (Day 15), with PC1- showing the greater elevation. In sebaceous units in telogen, PC1-IR was present at low levels in sebocytes and in adjacent small cells, whereas PC2-IR was at or below detectable levels. In anagen VI, IR for both convertases increased, with PC1-IR again showing the greater increase. Both sebocytes and adjacent small cells showed the increase in PC1-IR, whereas only small cells showed the increase in PC2-IR (Fig 6).
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Discussion |
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In this report we have described the spatial and temporal expression of POMC mRNA, the POMC-derived peptides ACTH, ß-endorphin, ß-MSH, and -MSH, and the prohormone converting enzymes PC1 and PC2 during depilation-induced anagen in the murine hair cycle. In anagen, during regeneration of the hair follicle, the expression of POMC mRNA exhibited tissue-specific variations that were consistent with POMC-derived peptide immunoreactivities. Cells that contained POMC peptide IR also expressed POMC mRNA, and where the IR increased there was a parallel increase in mRNA. The levels of PC1 and PC2-IR also showed cell-specific variations and were found in the same cells that expressed POMC peptides. When the cleavage specificities of these convertases are examined (
-MSH, it can be inferred that PC1 and PC2 activities are responsible for the cell-specific differential processing of POMC in murine skin.
We had previously shown by RT-PCR that POMC mRNA was constitutively expressed throughout the murine hair cycle, with significant upregulation during the growing phase of the cycle (
In other studies, POMC gene transcripts have been detected by Northern blotting analysis in isolated cell lines derived from skin: in cultured human keratinocytes (
Commensurate with the presence of POMC message in these skin compartments was immunoreactivity for one or more POMC-derived neuropeptides (summarized in Table 1). ACTH-IR and ß-MSH-IR were both present in epidermal and follicle keratinocytes. However, the appearance of ß-MSH-IR preceded that of ACTH-IR in epidermal keratinocytes and decreased to telogen levels sooner than ACTH in follicular keratinocytes. ß-Endorphin-IR was not detected in these cells. Immunoreactivities for ß-endorphin, ß-MSH, and -MSH were present in sebaceous units, but ACTH-IR was not. ß-Endorphin-IR was restricted to sebaceous units throughout the hair cycle and is consistent with previous reports on ß-endorphin-IR in skin (
As a first approximation in the identification of the processing enzymes that may be responsible for this differential expression of POMC peptides, our immunocytochemical data show that both PC1-IR and PC2-IR were present in keratinocytes at telogen and that both had increased by mid-anagen. PC1-IR was greater than PC2-IR and increased to a greater extent. In sebaceous units, on the other hand, PC1-IR was detected at low levels in sebocytes and adjacent small cells, whereas PC2-IR was at or below detectable levels. Both sebocytes and small cells showed an increase in PC1-IR, but only the small cells showed an increase in PC2-IR. In analogy with the pituitary, in cells in the skin, the differential expression of PC1 and PC2 may play a role in posttranslational POMC processing seen there (
The activity of PC1, alone or in combination with furin, another prohormone convertase that is ubiquitously expressed, could account for the processing of POMC to ACTH, ß-endorphin, and ß-MSH observed in skin, because (a) PC1 was present in all skin cells at all times examined and (b) PC1-IR increased with progression of anagen. Although the presence of furin has not been demonstrated in skin, it appears to be ubiquitously expressed in all of the cells and tissues examined (-MSH (
-MSH-IR, and both PC1-IR and PC2-IR increased in sebaceous units with progression of anagen. Therefore, the increase in PC2 could account for the increase in ß-endorphin-IR. Similarly, the dramatic increase in ß-MSH-IR, which peaked at mid-anagen and fell back to telogen levels by late anagen, could also have resulted from this increase in PC2.
The prohormone convertases PC1 and PC2 are considered by many to be restricted in their expression to neuroendocrine cells (
Do keratinocytes possess a regulated secretory pathway? The upward migration of keratinocytes in the epidermis from the stratum basal through the stratum spinosum and stratum granulosum to the stratum corneum is accompanied by differentiation of the keratinocyte into a secretory granular cell. These cells are responsible for the biogenesis of lamellar bodies (LBs) or lamellar granules, the contents of which, when secreted, are responsible for the formation of the epidermal permeability barrier (
In summary, we have demonstrated hair cycle-associated changes in spatial and temporal expression of the convertases PC1 and PC2 and of ACTH, -MSH, ß-MSH, and ß-endorphin peptides in murine skin, and we suggest that they are the result of both POMC mRNA translation and cleavage specificities of PC1 and PC2 enzymes.
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Acknowledgments |
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Supported in part by grants from the NSF: IBN 9405242 and IBN 9604354 to AS and JEM, and from the NIH: S10RR12894 to JEM.
We thank Drs B.A. Eipper (Johns Hopkins University School of Medicine), R.G. Allen (Oregon Health Sciences Center), and S.H. Pomerantz (University of Maryland) for gifts of antisera, Drs G. Davila and J. Pawelek (Yale University) for gifts of POMC peptides, and Dr J.L. Roberts (Mount Sinai Medical Center) for the mouse POMC cDNA. We acknowledge the Imaging Core Facility at the Albany Medical College (J.E. Mazurkiewicz, director) for use of the confocal microscope. We also thank Dr G. Ermak (University of Southern California) for expert technical assistance and discussion in aspects of the molecular biology.
Received for publication December 30, 1999; accepted March 15, 2000.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Allen RG, Herbert E, Hinman M, Shihuta H, Pert CB (1978) Coordinate control of corticotropin, ß-lipotropin, and ß-endorphin release in pituitary cell cultures. Proc Natl Acad Sci USA 75:4972-4976[Abstract]
Allen RG, Orwoll E, Kendall JW, Herbert E, Paxton M (1980) The distribution of forms of adrenocorticotropin and beta-endorphin in normal, tumorous, and autopsy human pituitary tissues: virtual absence of 13K adrenocorticotropin. J Clin Endocrinol Metab 51:376-379[Abstract]
Ances G, Pomerantz SH (1974) Serum concentrations of ß-melanocyte-stimulating hormone in human pregnancy. Am J Obstet Gynecol 119:1062-1068[Medline]
Arvan P, Castle D (1998) Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem J 332:593-610[Medline]
Autelitano DJ, Lundblad JR, Blum M, Roberts JL (1989) Hormonal regulation of POMC gene expression. Annu Rev Physiol 51:715-726[Medline]
Benjannet S, Rondeau N, Day R, Chretien M, Seidah NG (1991) PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 88:3564-3568[Abstract]
Can G, Abdel-Malek Z, PorterGill PA, Gill P, Boyce S, Grabowski GA, Nordlund J, Farooqui J (1998) Identification and sequencing of a putative variant of proopiomelanocortin in human epidermis and epidermal cells in culture. J Invest Dermatol 111:485-491[Abstract]
Day R, Schafer MK, Watson SJ, Chrétien M, Seidah NG (1992) Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. Mol Endocrinol 6:485-497[Abstract]
DeBault LE, Wang B (1994) Localization of mRNA by in situ transcription and immunogold-silver staining. Cell Vis 1:67-70
Elias PM, Cullander C, Mauro T, Rassner U, Kömüves L, Brown BE, Menon GK (1998) The secretory granular cell: the outermost granular cell as a specialized secretory cell. J Invest Dermatol Symp Proc 3:87-100
Ermak G, Slominski A (1997) Production of POMC, CRH-R1, MC1, and MC2 receptor mRNA and expression of tyrosinase gene in relation to hair cycle and dexamethasone treatment in the C57BL/6 mouse skin. J Invest Dermatol 108:160-165[Abstract]
Farooqui JZ, Medrano EE, Abdel-Malek Z, Nordlund J (1993) The expression of proopiomelanocortin and various POMC-derived peptides in mouse and human skin. Ann NY Acad Sci 680:508-510[Medline]
Furkert J, Klug U, Slominski A, Eichmuller S, Mehlis B, Kertscher U, Paus R (1997) Identification and measurement of beta-endorphin levels in the skin during induced hair growth in mice. Biochim Biophys Acta 1336:315-322[Medline]
Hatsuzawa K, Hosaka M, Nakagawa T, Nagase M, Shoda A, Murakami K, Nakayama K (1990) Structure and expression of mouse furin, a yeast Kex2-related protease. Lack of processing of coexpressed prorenin in GH4C1 cells. J Biol Chem 265:22075-22078
Hook VY, Azaryan AV, Hwang SR, Tezapsidis N (1994) Proteases and the emerging role of protease inhibitors in prohormone processing. FASEB J 8:1269-1278
Kuliawat R, Klumperman J, Ludwig T, Arvan P (1997) Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells. J Cell Biol 137:595-608
Luger TA, Scholzen T, Brzoska T, Becher E, Slominski A, Paus R (1998) Cutaneous immunomodulation and coordination of skin stress responses by alpha-melanocyte-stimulating hormone. Ann NY Acad Sci 840:381-394
Madison KC, Sando GN, Howard EJ, True CA, Gilbert D, Swartzendruber DC, Wertz PW (1998) Lamellar granule biogenesis: a role for ceramide glucosyltransferase, lysosomal enzyme transport, and the Golgi. J Invest Dermatol Symp Proc 3:80-86
Marcinkiewicz M, Day R, Seidah NG, Chrétien M (1993) Ontogeny of the prohormone convertases PC1 and PC2 in the mouse hypophysis and their colocalization with corticotropin and alpha-melanotropin. Proc Natl Acad Sci USA 90:4922-4926[Abstract]
Menon GK, Feingold KR, Elias PM (1992) Lamellar body secretory response to barrier disruption. J Invest Dermatol 98:279-289[Abstract]
Muller L, Zhu X, Lindberg I (1997) Mechanism of the facilitation of PC2 maturation by 7B2: involvement in ProPC2 transport and activation but not folding. J Cell Biol 139:625-638
PanoskaltsisMortari A, Bucy RP (1995) In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts. BioTechniques 18:300-307[Medline]
Paus R, Eichmuller S, van der Veen C, Kopp T, Hagen E, MullerRover S, Hofmann U (1998) Generation and cyclic remodeling of the hair follicle immune system in mice. J Invest Dermatol 111:7-18[Abstract]
Seidah NG, Day R, Marcinkiewicz M, Chrétien M (1993) Mammalian paired basic amino acid convertases of prohormones and proproteins. Ann NY Acad Sci 680:135-146[Medline]
Seidah NG, Gaspar L, Mion P, Marcinkiewicz M, Mbikay M, Chrétien M (1990) cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases DNA. Cell Biol 9:415-424
Seidah NG, Marcinkiewicz M, Benjannet S, Gaspar L, Beaubien G, Mattei MG, Lazure C, Mbikay M, Chrétien M (1991) Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol 5:111-122[Abstract]
Simmons DM, Arriza JL, Swanson LW (1989) A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes. J Histotechnol 12:169-181
Slominski A, Botchkareva NV, Botchkarev VA, Chakraborty A, Luger T, Uenalan M, Paus R (1998) Hair cycle-dependent production of ACTH in mouse skin. Biochim Biophys Acta 1448:147-152[Medline]
Slominski A, Ermak G, Hwang J, Mazurkiewicz JE, Corliss D, Eastman A (1996) The expression of proopiomelanocortin (POMC) and of corticotropin releasing hormone receptor (CRH-R) genes in mouse skin. Biochim Biophys Acta 1289:247-251[Medline]
Slominski A, Mihm MC (1996) Potential mechanism of skin response to stress. Int J Dermatol 35:849-851[Medline]
Slominski A, Paus R, Mazurkiewicz JE (1992) Proopiomelanocortin expression in the skin during induced hair growth in mice. Experientia 48:50-54[Medline]
Slominski A, Wortsman J, Mazurkiewicz JE, Matsuoka L, Dietrich J, Lawrence K, Gorbani A, Paus R (1993) Detection of proopiomelanocortin-derived antigens in normal and pathologic human skin. J Lab Clin Med 122:658-666[Medline]
Smeekens SP, Avruch AS, LaMendola J, Chan SJ, Steiner DF (1991) Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc Natl Acad Sci USA 88:340-344[Abstract]
Smeekens SP, Montag AG, Thomas G, AlbigesRizo C, Carroll R, Benig M, Phillips LA, Martin S, Ohagi S, Gardner P (1992) Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3. Proc Natl Acad Sci USA 89:8822-8826[Abstract]
Smith AI, Funder JW (1988) Proopiomelanocortin processing in the pituitary, central nervous system, and peripheral tissues. Endocrine Rev 9:159-179[Medline]
Thomas L, Leduc R, Thorne BA, Smeekens SP, Steiner DF, Thomas G (1991) Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 88:5297-5301[Abstract]
Wakamatsu K, Graham A, Cook D, Thody AJ (1997) Characterisation of ACTH peptides in human skin and their activation of the melanocortin-1 receptor. Pigment Cell Res 10:288-297[Medline]
Whitfeld PL, Seeburg PH, Shine J (1982) The human pro-opiomelanocortin gene: organization, sequence, and interspersion with repetitive DNA. DNA 1:133-143[Medline]
Wintzen M, Gilchrest BA (1996) Proopiomelanocortin, its derived peptides, and the skin. J Invest Dermatol 106:3-10[Abstract]
Wintzen M, Yaar M, Burbach JP, Gilchrest BA (1996) Proopiomelanocortin gene product regulation in keratinocytes. J Invest Dermatol 106:673-678[Abstract]
Zhou A, Bloomquist BT, Mains RE (1993) The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing. J Biol Chem 268:1763-1769
Zhou A, Mains RE (1994) Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2. J Biol Chem 269:17440-17447