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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 10 3582-3588
Copyright © 2000 by The Endocrine Society

Original Studies

Liquid Chromatography-Mass Spectrometry Detection of Corticotropin-Releasing Hormone and Proopiomelanocortin-Derived Peptides in Human Skin1

Andrzej Slominski, Andrzej Szczesniewski and Jacobo Wortsman

Department of Pathology (A.Sl.), University of Tennessee, Memphis, Tennessee 38163; Hitachi Scientific Instruments, Inc. (A.Sz.), Naperville, Illinois; and Department of Internal Medicine (J.W.), Southern Illinois University, Springfield, Illinois 62794

Address correspondence and requests for reprints to: Dr. Andrzej Slominski, Department of Pathology, RM576-BMH Main, University of Tennessee, 899 Madison Avenue, Memphis, Tennessee 38163. E-mail: aslominski{at}utmem.edu


   Abstract
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 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We have previously shown expression of CRH and POMC genes and peptides in the human skin. To ascertain the identity of those peptides, we used methods of peptide extraction and purification combined with the highly specific technique of liquid chromatography-mass spectrometry. Testing extracts of human skin, we identified endogenous peptides with masses and retention times corresponding to CRH, ACTH 1–39, ACTH 1–13, and {alpha}-MSH standards. Thus, conclusive evidence is provided for the presence of CRH and the POMC-derived ACTH 1–39, ACTH 1–13, and {alpha}-MSH peptides in human skin. Direct identification of these peptides is consistent with translation of the corresponding genes, and it also suggests intermediate pituitary lobe-like POMC peptide processing.


   Introduction
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 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
WE HAVE proposed that skin defense against environmental stimuli is regulated by a local CRH/POMC-mediated pathway(s) that would become operative within the context of a stress response system (1, 2, 3, 4). This concept is based on the well-known characteristic of the skin of being a significant target for ACTH and MSH peptides (2, 4, 5, 6, 7, 8, 9) and on recent data showing that the phenotype of skin cells can be regulated by CRH and urocortin through interactions with their specific receptors (3, 9, 10, 11, 12, 13, 14, 15, 16). Full expression of such a system at the local level has been confirmed by the capability of skin itself to express the POMC, CRH, and urocortin genes and to translate the transcription products into the corresponding proteins (2. 4, 6–9, 17–21). The actual detection of POMC-derived {alpha}-MSH, ACTH and ß-endorphin peptides, and CRH in skin was previously performed with methods of RP-HPC separation combined with specific RIA (7, 21, 22). However, more definitive verification was deemed necessary, because {alpha}-MSH and ACTH are known to exist in different forms (5), and CRH is a member of larger family of related peptides (23, 24) that may include species that remain to be identified. Therefore, we ascertained the identity of the {alpha}-MSH and CRH species in human skin using methods of peptides extraction and purification combined with the highly specific technique of liquid chromatography-mass spectrometry (LC-MS) (20).


   Subjects and Methods
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 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Materials

Fresh human skin was obtained from tissue removed during surgery performed on burn patients. Grossly noninvolved (perilesional) skin was separated, frozen quickly in liquid nitrogen, and stored at -80 C until the time of analysis. The use of human tissues was approved by the Loyola University Medical Center Committee on Research Involving Human Subjects (protocol: skin as neuroendocrine organ, exempt., cat. 4). ACTH 1–39, ACTH 1–24, ACTH 1–17, ACTH 1–13, {alpha}-MSH, and diacetyl-{alpha}-MSH standards were purchased from Sigma (St. Louis, MO). Synthetic human CRH peptide was purchased from a commercial supplier (Molecular Research Laboratory, Durham, NC).

Peptide extraction

Human skin, while still frozen under liquid nitrogen, was pulverized in a mortar with a pestle and then extracted in acetonitrile/H2O (1/1) (19, 20). Tissue extracts were centrifuged at 30,000 x g for 30 min at 4 C, the supernatants were collected and extracted with pentane (1/25 vol of sample), and the resulting extracts were evaporated in speed-vac (19, 20). These evaporates were resuspended in 0.5% Triton X-100 in PBS, pH 7.4, containing 1 mmol/L phenylmethylsulfonylfluoride and 0.01% aprotinin, and centrifuged at 16,000 x g; the supernatants were combined with equal amounts of 0.1% trifluoroacetic acid (TFA) and passed through a SEPCOL-1 columns (18, 19, 20, 21). After three washes with 0.1% TFA (5 mL each), the peptides were eluted with acetonitrile (20%), and the eluates were evaporated in speed-vac before storage at -80 C until analysis.

LC-MS

Actual identification of CRH, {alpha}-MSH, and ACTH peptides was accomplished with LC-MS using model M-8000 LC/3DQ-MS quadropole ion trap mass spectrometer (Hitachi Scientific Instruments, Inc., San Jose, CA), in the tandem mode (20). Briefly, the evaporated eluates from purification by SEPCOL-1 (see above) were dissolved in 0.1% TFA, sonicated, and centrifuged in a Marathon 13K microfuge at 2000 rpm for 5 min. The supernatants were then filtered through MSI MAGNA nylon Cameo filters (0.45-µm pore size) and separated by RP-HPLC on a 7000 System (Hitachi). For mass determination, the experimental samples and peptide standards were dissolved in 0.1% TFA and separated through a C18 Vydac column 218TP52 (250 x 2.1 mm; particle size, 5 µm) (Vydac/The Separations Group, Hesperia, CA) with a mobile phase A: 0.5% or 0.1% TFA, for the identification of CRH, {alpha}-MSH, and ACTH peptides. Separation was performed with acetonitrile (mobile phase B) at the following gradients: 5% (0–5 min), 5–40% (5–60 min), 40% (60–70 min), 40–80% (70–80 min), 80% (80–90 min), while the flow rate was maintained at 0.25 mL/min. The effluent from the HPLC system was routed to the MS through electrospray interphase (ESI). The ESI conditions were as follow: gas temp:150 C; desolvator temp, 200 C; aperture 1 temp, 170 C; aperture 2 temp, 120 C; focus voltage, 4 kV; drift voltage, 60 V; and focus voltage, 30 V.


   Results
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 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The results of experimental determination of elution time and actual mass of the CRH standard by the tandem technique of LC-MS are shown in Fig. 1Go. When using 0.5% TFA as mobile phase, and increasing concentrations of acetonitrile, the CRH standard eluted from the C18 column at 65–66 min and, after routing to the MS with ESI, it showed average multiple charge (+4) mass spectrum (M+4H)4+ ion at m/z 1189. This resulted in a calculated molecular mass of 4752 Da. The calculated mass of the CRH standard is shifted to the left by 5 Da (expected value, 4757 Da ) because of the calibration settings used during testing. Human skin extracts, processed with LC-MS under the same conditions, showed a peptide with identical mass spectrum (M+4H)4+ ion at m/z 1189 (Fig. 1AGo), and retention time at 65–66 min (Fig. 1BGo), as the CRH standard (inserts). Thus, we conclude that CRH peptide is present in human skin, in agreement with our previous identification of CRH immunoreactivity by methods of RP-HPLC and RIA (18, 19).



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  		Figure 1. Identification of CRH, in human skin, by LC/MS. A, Endogenous peptide (CRH) with the same retention time of 65–66 min as the CRH standard (inset). B, The endogenous peptide (CRH) also has the same multiple charge (+4) mass as the CRH standard (inset; calculated mass, 4752 Da).

 
The mass spectrum (M+2H)2+ ion of the ACTH 1–13 standard occurred at m/z 812 (calculated mass, 1622 Da ), eluting from the C18 column at 40 min, and the mass spectrum (M+2H)2+ ion of {alpha}-MSH occurred at m/z 832.5 (calculated mass, 1663 Da ), eluting from the column at 41 min (Fig. 2Go; insets). The calculated molecular masses, under experimental conditions, were very similar to the theoretical values of 1624 and 1665 Da for ACTH 1–13 and {alpha}-MSH, respectively. Human skin extracts, processed under the same conditions, showed peptides identified as ACTH 1–13 and {alpha}-MSH, because they had mass spectrum (M+2H)2+ ions at m/z 812 (Fig. 2AGo) and retention time (40 min) (Fig. 2BGo) and at m/z 832.4 (Fig. 2CGo) and retention time (41 min) (Fig. 2DGo), identical to the corresponding standards. We made a special attempt at identifying, in the skin extracts, the presence of mass spectra corresponding to and eluting with the same retention time as the standards for ACTH 1–39 (49 min), ACTH 1–24 (40 min), ACTH 1–17 (36 min), and diacetyl-{alpha}-MSH (43.5 min). We could then detect a peptide with the same mass spectrum (M+4H) 4+ ion at m/z 1135.8 (calculated mass of 4540 Da; theoretical value of 4541 Da) and retention time as the ACTH 1–39 standard, at a level slightly above background (Fig. 3Go). This result documents the presence, in the skin, of a trace amount of full-length ACTH. The peak masses corresponding to the ACTH 1–24, ACTH 1–17, and diacetyl-{alpha}-MSH standards were buried within the background (noise level), which precluded definitive identification of the corresponding peptides (data not shown).




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  		Figure 2. Identification of ACTH 1–13 and {alpha}-MSH, in human skin, by LC/MS. A, Endogenous peptide with the same multiple charge (+2) mass as the ACTH 1–13 standard (inset; calculated mass, 1622 Da). B, Endogenous peptide in human skin extracts also has the same retention time of 40 min as the ACTH 1–13 standard (inset). C, Endogenous peptide with the same multiple charge (+2) mass as the {alpha}-MSH standard (inset; calculated mass, 1663 Da). D, Endogenous peptide in human skin extracts also has the same retention time of 41 min as the {alpha}-MSH standard (inset).

 


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  		Figure 3. Identification of ACTH 1–39, in human skin, by LC/MS. A, Endogenous peptide (ACTH) with the same multiple charge (+4) mass (calculated molecular mass, 4540 Da) as the ACT 1–39 standard. B, The peptide also elutes at the same retention time (49 min) as the ACTH 1–39 standard.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In previous studies that employed methods of RP-HPLC with RIA, the presence of {alpha}-MSH in mammalian skin was demonstrated in human epidermis and whole human skin (7, 21, 22). Full-length ACTH (1–39) was also detected in human skin cells (7, 21), together with products of its processing, such as ACTH 1–17, diacetyl-{alpha}-MSH, ACTH 1–10, and acetyl ACTH 1–10 (7). In the present work, we could definitively identify {alpha}-MSH and ACTH 1–13, by LC-MS, in the skin of burn patients; and we obtained data showing the presence of a trace amount of full-length ACTH. However, processing products that included ACTH 1–17 and diacetyl-{alpha}-MSH were below the limits of detectability, which was estimated to be 1–5 pmols for {alpha}-MSH standard, per ESI MS determination (not shown). In this context, the comparatively higher concentration of {alpha}-MSH and of ACTH 1–13 in perilesional skin may be attributable to their accumulation in response to extreme cutaneous stress (thermal burn)(4). Though the role of {alpha}-MSH in melanogenesis has been clarified (5, 25), and its action in counteracting noxious stimuli is being elucidated (2, 4, 8, 9), that of ACTH 1–13 at this comparatively high concentration remains a subject of speculative analysis. For example, it could serve a biological function in rapid regulation of homeostasis in damaged skin, through interactions with MC-1 (5, 7, 25), and possibly MC-2, receptors (26, 27). Alternatively, it could be only an inactive end product of rapid degradation of ACTH 1–39 without any biological function.

The detection of ACTH 1–13 and {alpha}-MSH cannot be equated with local synthesis; nevertheless, the comparatively very low levels of the ACTH 1–39 precursor would suggest a local source from sequential processing of POMC, rather than uptake of circulating ACTH. This melanotropic routing of POMC processing, operating in the skin, would then be analogous to that in the intermediate pituitary lobe (5, 28, 29). Such a processing mechanism could provide the explanation for a common clinical observation. Thus, the known activation of the POMC-MC receptor system by local stress such as trauma or solar radiation (containing ultraviolet light spectrum) (4, 30) may also mediate the localized skin pigmentation in the stress-affected areas of patients with Addison disease. In this setting, the extremely high prevailing plasma ACTH levels (29) could overcome the dermal-epidermal barrier (4) and, after reaching keratinocytes and melanocytes, ACTH could be further processed to {alpha}-MSH. Together with stress-induced expression of MC receptors, and processing machinery with generation of {alpha}-MSH, this would result in production of the typical pattern of cutaneous pigmentation in the sun-exposed areas associated with primary adrenal failure.

In summary, using LC/MS, we have provided conclusive evidence for the presence of CRH, ACTH 1–39, ACTH 1–13, and {alpha}-MSH in the human skin.


   Footnotes
 
1 Supported by grants (to A.S.) from the National Science Foundation (IBN-9896030), American Cancer Society, Illinois Division (no. 99–51), and from Banes Charitable Foundation (LU no. 9178). Back

Received April 4, 1999.

Revised June 13, 2000.

Accepted July 7, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Slominski A, Mihm M. 1996 Potential mechanism of skin response to stress. Int J Dermatol. 35:849–851.[Medline]
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  3. Slominski A, Botchkarev V, Choudhry M, et al. 1999 Cutaneous expression of CRH and CRH-R: is there a "skin stress system"? Ann NY Acad Sci. 885:287–311.[Abstract/Free Full Text]
  4. Slominski A, Wortsman J, Paus R, Luger T, Salomon S. 2000 Corticotropin-releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. Physiol Rev. 80:979–1020.[Abstract/Free Full Text]
  5. Eberle AN. 1988 The melanotropins: chemistry, physiology and mechanism of action. New York: S. Karger Publ.
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  14. Singh LK, Boucher W, Pang X, et al. 1999 Potent mast cell degranulation and vascular permeability triggered by urocortin through activation of corticotropin releasing hormone receptors. J Pharmacol Exp Ther. 288:1349–1356.[Abstract/Free Full Text]
  15. Fleisher-Berkovich S, Danon A. 1995 Effect of corticotropin-releasing factor on prostaglandin synthesis in endothelial cells and fibroblasts. Endocrinology. 136:4068–4072.[Abstract]
  16. Slominski A, Roloff B, Zbytek B, et al. 2000 Corticotropin releasing hormone (CRH) and related peptides can act as bioregulatory factors in human keratinocytes. In Vitro Cell Dev Biol. 36:211–216.[CrossRef]
  17. Slominski A, Ermak G, Hwang J, Mazurkiewicz J, 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]
  18. Slominski A, Ermak G, Mazurkiewicz JE, Baker J, Wortsman J. 1998 Characterization of corticotropin-releasing hormone (CRH) in human skin. J Clin Endocrinol Metab. 83:1020–1024.[Abstract/Free Full Text]
  19. Roloff B, Fechner K, Slominski A, et al. 1998 Hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors (CRF-R) in murine skin. FASEB J. 12:287–297.[Abstract/Free Full Text]
  20. Slominski A, Roloff B, Curry J, Dahiya M, Szczesniewski A, Wortsman J. 2000 The skin produces urocortin. J Clin Endocrinol Metab. 85:815–823.[Abstract/Free Full Text]
  21. Slominski A, Heasley D, Ermak G, Mazurkiewicz JE, Baker J, Carlson A. 1999 Expression of proopiomelanocortin (POMC) derived melanocyte stimulating hormone (MSH) and ACTH peptides in skin of basal cell carcinoma patients. Hum Pathol. l30:208–215.
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