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Original Studies |
Department of Pathology, Loyola University Medical Center (A.Sl., B.R., J.C., M.D.), Maywood, Illinois 60153; Hitachi Instruments, Inc. (A.Sz.), Naperville, Illinois; and the Department of Internal Medicine, Southern Illinois University (J.W.), Springfield, Illinois
Address all correspondence and requests for reprints to: Andrzej Slominski, M.D., Ph.D., Department of Pathology, Loyola University Medical Center, 2160 First South Avenue, Maywood, Illinois 60153. E-mail: aslomin{at}wpo.it.luc.edu
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Abstract |
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Introduction |
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Urocortin is a recently described member of the family of structurally related CRH-like peptides that share a high degree of homology (15, 16). In the case of the mammalian urocortin, its gene, similar to that of CRH, is composed of one intron and two exons, with exon 2 encoding the urocortin peptide (17). In humans, the final product of the urocortin gene is a peptide 40 amino acids long, with 45% homology to rat/human CRH and more than 90% homology with rat and mouse urocortin peptide (15, 16, 17). In rat brain, urocortin is particularly detectable in the Edinger-Westphal nucleus, lateral superior olive, substantia nigra, ventral tegmental area, linear and dorsal raphe nuclei, and hypothalamus (18, 19). In human brain, urocortin is more widespread, being found in every region tested, with the highest concentrations in the frontal cortex, temporal cortex, and hypothalamus; interestingly, it is also detected in the anterior pituitary gland (20, 21). Nevertheless, urocortin is also produced in various peripheral tissues such as placenta, uterus, immune system, stomach, small and large bowel, pancreas, adrenal gland, testis, and heart (22, 23, 24, 25). As urocortin acts as a ligand for the CRH receptors, CRH-R1 and CRH-R2 (26, 27, 28, 29), and these receptors are functionally expressed in the skin (3, 11, 12, 13, 14), we investigated the presence of the peptide in this organ.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human tissue. Fresh human skin that was used for molecular and biochemical analyses had been obtained as discards from surgical procedures performed on burn patients; human pituitaries were obtained from the National Hormone and Pituitary Program, NIDDK. Tissue specimens were stored at -80 C until the time of analysis. Samples of the biopsies from previously diagnosed lesional and nonlesional specimens were cut from paraffin-embedded blocks and used for immunocytochemical analyses. The use of human tissues was approved by the Loyola University Medical Center committee on research involving human subjects (protocol: skin as neuroendocrine organ, exemption, catalogue 4).
Animal tissue. C57BL/6 strain female mice (7 weeks old) and Syrian hamsters (males, 3 months old) were purchased from Taconic Farms, Inc. (Germantown, NY) and housed in community cages at the animal facilities of the Albany Medical College (Albany, NY). Active hair growth (anagen) was induced in the back skin of 8-week old C57BL/6 female mice following protocols routinely used in our laboratory (9, 30). The line of Bomirski hamster MI melanotic transplantable melanoma was propagated in male Syrian hamsters as previously described (31). Tissue specimens of skin, pituitary, and brain from hamsters and mice were collected as previously described and frozen rapidly in liquid nitrogen, and the tissues were stored at -80 C until further analysis, for up to 5 yr (9, 30, 31, 32). The experimental protocol was originally approved by the institutional animal care and use committee at Albany Medical College, and a similar protocol for mice was approved at Loyola University Medical Center.
Cell cultures. Human keratinocytes (HaCaT) were propagated in DMEM, whereas squamous cell carcinoma (C4–1), human melanoma (SK-MEL188), and hamster melanoma (Bomirski AbC-1) cells were grown in Ham’s F-10 medium as described previously; the media were supplemented with 10% FBS and antibiotics (9, 11, 33). Conditions for culture of human keratinocytes were described by Chakraborty et al. (6).
Methods
Peptide extraction. Frozen pellets of cultured cells and mouse skin specimens were extracted in 0.5% Triton X-100 in phosphate-buffered saline, pH 7.4 (sample buffer), containing 1 mmol/L phenylmethylsulfonylfluoride and 0.01% aprotinin and centrifuged at 16,000 x g; the resulting supernatants were combined with an equal amount of 0.1% of trifluoroacetic acid (TFA) and purified throughout SEPCOL-1 columns (9, 11, 12). Human and rodent skin were pulverized in a mortar with a pestle while still frozen under liquid nitrogen and then extracted in acetonitrile/H2O (1:1) (12). Tissue extracts were centrifuged at 30,000 x g for 30 min at 4 C, and the supernatants were collected and extracted with pentane (0.25 vol sample); the extracts were evaporated in a Speed-Vac (Savant Instrument Co., Farmingdale, NY) (12). The resulting evaporates were resuspended in sample buffer, centrifuged at 16,000 x g, combined with 0.1% TFA, and purified through SEPCOL-1 (see above). The eluates were collected, evaporated, and stored at -80 C until analysis.
Reverse phase high performance liquid chromatography (RP-HPLC). The peptides extracted from skin and cultured cells were separated by RP-HPLC on a Hitachi HPLC system (programmable low pressure gradient pump L 6000 A, autosampler AS 4000, DAD detector L 4500, HPLC software manager D 6000), using a Beckman Coulter, Inc., Ultrasphere C18 IP HPLC column; 150 x 4.6 mm; particle size, 5 µm). Mobile phase A was 0.1% TFA in water, and mobile phase B was 100% acetonitrile. Separation was performed at ambient temperature using a linear gradient of 20–50% acetonitrile for 45 min, with the flow rate set at 1 mL/min. The eluted fractions, collected at 1-min intervals, were evaporated. The elution time for synthetic urocortin peptide (gift from Dr. E. Wei) was determined in a separate HPLC column.
RIA. Evaporated samples of skin or cultured cells were prepared as described above, reconstituted in buffer, and assayed for urocortin using a RIA kit (Phoenix Pharmaceuticals, Inc., Mountain View, CA) according to the manufacturer’s protocol. The antibody did not separate human from rat urocortin (cross-reactivity of 100%). The cross-reactivity of cortistatin with urocortin was 1%. The antibody did not recognize CRH (human/rat), LH-RH or somatostatin.
Liquid chromatography-mass spectrometry (LC-MS). Actual identification of urocortin was accomplished with LC-MS using a model M-8000 LC/3DQ-MS quadropole ion trap mass spectrometer (Hitachi Instruments, Inc., San Jose, CA), in the tandem mode. Briefly, human skin extracts were purified by SEPCOL-1 (see above), dissolved in 0.1% TFA, sonicated, and centrifuged in a Marathon 13K microfuge at 2000 rpm for 5 min. The supernatant was then filtered through MSI MAGNA nylon Cameo filters (0.45 µm pore size) and separated by RP-HPLC on a Hitachi 7000 system. For mass determination, two gradient HPLC separation conditions were tested. In one of them (first condition) the samples were separated using a Zorbax HPLC column SB-C18 (150 x 2.1 mm; particle size, 5 µm; Hewlett-Packard Co., Chadds Fords, PA); mobile phase A was 0.1% TFA in water, and mobile phase B was 100% acetonitrile. Separation was performed at room temperature with a flow rate of 0.2 mL/min, using 100% phase A for 5 min, followed by a linear gradient of 0–50% phase B for 90 min. In the second condition the samples were dissolved in 0.1% TFA and separated using 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 of 0.5% TFA. Separation was performed using acetonitrile at the gradients 5% (0–5 min), 5–40% (5–60 min), 40% (60–70 min), and 40–80% (70–80 min), while maintaining the flow rate at 0.25 mL/min. The effluent from the HPLC system was routed to the MS through electrospray interphase. The electrospray interphase conditions were as follows: gas temperature, 150 C; desolvator temperature, 200 C; aperture 1 temperature, 170 C; aperture 2 temperature, 120 C; focus voltage, 4 kV; drift voltage, 60 V; and focus voltage 30 V.
RT-PCR amplifications. Total ribonucleic acid (RNA) was extracted from cells with the RNAzol B isolation solution (CINNA/BIOTECX Laboratories, Houston, TX) from human skin, human pituitary and adrenal glands, cultured keratinocytes, and squamous cell carcinoma melanoma cells (8, 9, 10, 11). Two micrograms of total RNA from each specimen were reverse transcribed using oligo(deoxythymidine) as primers and the Superscript preamplification system (Life Technologies, Inc., Gaithersburg, MD). To ensure that the samples were free of DNA contamination, PCR amplification tests were performed without prior RT, using primers for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (6, 8, 9, 10, 11, 34). Amplification of the 145-bp fragment representative of the exon 2 of human urocortin gene was performed by RT-PCR using primers and conditions described by Bamberger et al. (22). The cloned fragments from skin and HaCaT keratinocytes were cut from the gels and sent for sequencing to Commonwealth Biotechnologies, Inc. (Richmond, VA). Nucleotide sequencing was performed with the BigDye kit and a 377XL DNA sequencer from Perkin-Elmer Corp./PE Applied Biosystems (Commonwealth Biotechnologies, Inc.). To detect expression of the mouse urocortin gene we used primers and conditions designed by Dr. T. Kishimito. A 204-bp fragment from exon 2 was amplified using the following sequences: 5'-ACTGTCCATCGACCTCACCTTCCA-3' (sense primer) and 5'-ACTGAGACAGCTCCGGTTGTGC-3' (antisense primer). The reaction mixture contained 1.5 mmol/L MgCl2, 0.4 mmol/L deoxy-NTP, 0.2 mmol/L of upper and lower primers, and 20 mmol/L Tris-HCl, pH 8.4. The murine complementary DNA (cDNA), preheated at 95 C for 2 min, was amplified through 35 cycles of 15 s at 95 C, 30 s at 55 C, and 60 s at 72 C, with a final extension of 10 min at 72 C. The resulting PCR products were separated electrophoretically on 1% agarose gels, stained with ethidium bromide, and photographed under UV light.
Immunocytochemistry. Immunocytochemistry was performed according to standard protocols previously described (10, 30, 34). Briefly, formalin-fixed sections were deparaffinized, and the rehydrated slides were blocked for 30 min with 5% nonfat dry milk in phosphate-buffered saline plus 0.1% Triton X-100 or Super Bock solution (ScyTek Laboratories, Logan, UT). Tissue sections were incubated with goat antibody against urocortin diluted at 1:50 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Incubation was performed overnight at 4 C in a humidified chamber; the tissue sections were then washed and further incubated for 60 min at room temperature with biotin-linked antigoat antibody diluted at 1:200. Final processing was accomplished with the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA), in which color development is attained with 3,3'-diaminobenzidine.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 145-bp fragment representative of exon 2 of the human
urocortin gene was detected by RT-PCR (35 cycles) in RNA isolated from
human pituitary and adrenal glands, normal facial skin, skin with basal
cell carcinoma, cultured keratinocytes, HaCaT keratinocytes, squamous
cell carcinoma cells, and melanoma cells (Fig. 1
, A and B). Sequencing of the cloned
fragment showed complete matching with the published urocortin cDNA
sequence (16), indicating transcription of the coding region of the
urocortin human gene. Similar testing, changing the set of primers to
detect the mouse urocortin gene, showed the 204-bp fragment
representative of exon 2 in the skin of the C57BL/6 mouse (Fig. 2).
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Urocortin IR was detected with a specific antiurocortin RIA in
cultured hamster AbC-1 and human SK-MEL188 melanoma cells, HaCaT
keratinocytes, and squamous cell carcinoma C4–1 cells (Table 1). Urocortin IR was also detected in
human and mouse skin and in transplantable Bomirski MI hamster melanoma
(Table 2). The highest levels of
urocortin antigen were detected in transplantable hamster MI melanoma,
followed by perilesional skin from burn patients and telogen skin from
C57BL6 mice. In mouse skin, the levels decreased gradually from those
found in telogens during progression of the hair cycle; the lowest
concentration was observed in late anagen VI (Table 2). In the hamster,
urocortin was below the level of detection in skin, although readily
detectable in the brain samples.
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There was a single RIA peak at the same retention time (47–48 min) as
the synthetic urocortin standard (Fig. 3A) in mouse skin (Fig. 3B) and
HaCaT keratinocytes (Fig. 3C).
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shows the results of mass
determination with the tandem technique of LC-MS. The mass spectrum of
urocortin standard (m/z at 1173.8) eluted at 84–85 min from HPLC and
was +4 ion (the calculated mass was 1173.8 x 4 = 4695 Da;
Fig. 4A). Human skin extracts processed with LC-MS under the same
conditions showed a peptide (URC) with identical +4 ion at m/z 1173.8
and retention time (84–85 min) as the urocortin standard (Fig. 4B).
Background subtraction further confirmed the presence of urocortin in
human skin (Fig. 4 C). These results were observed consistently using
different columns and HPLC gradients (data not shown). Nevertheless, it
must be noted that the calculated mass of the urocortin standard and
experimental results were shifted to the left by 12 Da (expected value,
4707 Da) due to the calibration setting used during this analysis,
which was verified after completion of the experiment.
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The urocortin antigen was localized to keratinocytes in epidermis
and the outer and inner root sheaths of hair follicles, epithelium of
sebaceous and eccrine glands, erector pili muscle, cutaneous blood
vessels walls, cutaneous nerves, and dermal mononuclear cells (Figs. 5 and 6).
Urocortin detected in pathological skin specimens was localized to
melanocytes of compound and junctional nevi (Fig. 7) as well as in melanoma cells, basal
cell carcinoma cells, and spindle cells of residual dermatofibrosarcoma
protuberans. Overall, the urocortin antigen was detected in 13 of 15
samples of perilesional and lesional skin. The presence of urocortin
was considered indeterminate, because of high stain background, in one
of the cases of perilesional burn skin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The evidence provided by immunocytochemistry combined with the studies in cultured cells strongly suggest that in human skin urocortin originates predominantly from the epithelial compartment. This is based on the detection of gene expression in cultured normal and malignant keratinocytes, and the finding of urocortin antigen localized to epidermal and follicular keratinocytes and sweat glands. Nonetheless, the detection of urocortin antigen in nevocytes and malignant melanocytes suggests that these cells are also capable of producing urocortin in vivo. As regards the detection of urocortin in other skin compartments such as blood vessels walls, dermal smooth muscle, mononuclear inflammatory cells, and dermal spindle cells, it could either represent CRH receptor-mediated peptide internalization or actual urocortin production by those cells. Definitive identification of an extraepithelial source(s) of the urocortin peptide will probably require detailed in situ hybridization studies.
We previously documented expression of the CRH gene in human skin with corresponding production of the peptide (3, 8, 9, 11). In contrast, mouse skin exhibited hair cycle-dependent presence of CRH peptide, but without expression of the CRH gene (10, 12). We then proposed that the source of CRH in mouse skin was local accumulation of CRH imported to the skin through afferent nerves (10, 12). In the present study both expression of urocortin gene and production of the peptide were detected in human and mouse skin. In the C57BL/6 mouse, the urocortin skin concentration was again related to the phase of the hair cycle, e.g. the highest concentration was found in telogen with decrease during anagen progression to the lowest levels in anagen VI. However, this cyclic pattern is the opposite of the variation in CRH content, which was lowest in telogen and increased during anagen development, to reach its highest level in anagen III–V (12). The significance of this asynchronicity in peptide expression may extend beyond that of a functional correlate of hair growth. Thus, in rodents it is not only the hair but the entire skin physiology that changes during progression from the resting phase (telogen) to the growing phase (anagen) (35, 36). These changes involve the formation of new miniorgans (hair bulbs), architectural development and pattern of epithelial cell proliferation and differentiation, the degree of functional activation of the local immune system, and the overall thickness of the skin with complementary vascularization and innervation (35, 36, 37). Melanogenic activity is also hair cycle dependent and starts in anagen III to reach its peak in anagen VI (35). Regardless of the role played by the local expression of urocortin in that complex sequence of events, it would have to be different from that played by CRH.
The precise action of urocortin in the regulation of peripheral organs function has not been defined completely and requires further experimental testing. However, it is recognized that urocortin can affect cellular phenotype through interaction with CRH receptors type 1 and 2 (26, 27, 28, 29). Acting as a ligand for these receptors, urocortin can inhibit trauma-induced edema (38); it can also induce placental ACTH and PG release (39), regulate vascular tone (26, 27, 28, 29), and protect cardiac myocytes from hypoxia-induced cell death (24). Furthermore, because of the presence of CRH-R2 in these tissues, urocortin has the potential to regulate skeletal and cardiac muscle contractility (26, 27, 28), and have also immunomodulatory activity (22). CRH peptide is a recognized proinflammatory agent (40). Both urocortin and CRH degranulate skin mast cells via the CRH-R1-mediated pathway, consistent with this proinflammatory action (41, 42). We have reported the detection of CRH-R1 gene expression in human and mouse skin (8, 9, 10, 11, 12) and, more recently, of the CRH-R2 gene in mouse skin (3). CRH-R gene expression was found to correlate with the actual presence of functional CRH receptors in skin and in a number of cultured cutaneous cells, including normal and malignant keratinocytes, fibroblasts, endothelial cells, and melanoma cells (3, 8, 9, 10, 11, 12, 13, 14, 43, 44). Thus, multiple potential targets for locally produced urocortin are present in the skin. This suggests that urocortin could act in the regulation of cutaneous function through auto- and/or paracrine mechanisms.
The placenta is a peripheral organ that has a similar ability to produce urocortin, CRH, and POMC peptides and to express functional receptors for those peptides; the local function of this system has also been partially elucidated (25, 39, 45, 46, 47, 48). Thus placenta, which is located at the interphase between fetal and maternal environment, uses variants of the classical hypothalamic and pituitary hormones to coordinate the response to stressful signals and modify local homeostasis for optimal maintenance of pregnancy and successful delivery (39, 45, 46, 47, 48). As the skin is similarly exposed to physical, chemical, and biological insults (1, 2, 3), the cutaneous neuroendocrine system composed of CRH-related molecules and POMC peptides, could operate in a manner comparable to that of placenta and gestational tissues. Thus, appropriate environmental signals that are continuously recognized and integrated in the skin could activate this system to rapidly counteract the damaging effect of noxious stimuli to reestablish tissue and systemic homeostasis (1, 2, 3). Therefore, urocortin production could be conceived as a general marker of stress exposure. It then becomes apparent that detection of this conserved neuropeptide signaling system in the skin indicates the need for further research on the local action of neuroendocrine factors. This may confirm the already established functional relationship between CRH-related molecules and phenotypic effects in placenta and gestational tissues.
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Acknowledgments |
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Footnotes |
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Received July 14, 1999.
Revised October 15, 1999.
Accepted October 20, 1999.
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References |
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-melanocyte-stimulating hormone. Ann NY Acad
Sci. 840:381–394.[CrossRef][Medline]
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