Expression of Diazepam-Binding Inhibitor Peptide in Human Skin: An Immunohistochemical and Ultrastructural Study

Hannu Alho, Annikki Vaalasti, Irina Podkletnova, and Leena Rechardt Department of Biomedical Sciences, University of Tampere and Department of Dermatology, Tampere University Hospital, Tampere, Finland

Human diazepam binding inhibitor (OBI) was originally isolated from the brain and subsequently found to be present in several peripheral tissues. The various physiologic effects recently attributed to OBI include acting as an endogenous ligand for the central and peripheral (mitochondrial) benzo­diazepine receptors. The present work provides, for the first time, evidence of OBI immunoreactivity in skin. OBI im­munoreactivity was found in the epidermis, in the eccrine sweat and in sebaceous glands. Ultrastructurally, OBI was

Although benzodiazepines are in extensive clinical use for their well-known anxiolytic, hypnotic, and anti­convulsant activities, recent experimental evidence has shown that they may also have other functions, such as the regulation of steroid synthesis, neoplastic

growth, and the immune response [1-3]. Whereas their clinical use is based on a central nervous system action mediated through the classic central benzodiazepine receptor (CBR) sites located on the GABA" receptor complex, the experiments concerned are based on interaction with a second class of benzodiazepine receptors, called peripheral or mitochondrial benzodiazepine receptors (MER) [4,5]. MERs playa central role in the regulation of lipid and steroid synthesis and are widely distributed in peripheral tissues including skin [6,7] .

Recently an endogenous polypeptide capable of displacing ben­zodiazepine binding to both CBR and MER has been purified from the brain and liver of different species and designated as diazepam binding inhibitor (DBI) (see [8,9]). Physiologic effects recently at­tributed to DEI include regulation of GABA" and MER receptors [10,11], binding to acyl-coenzyme A (acyl-CoA) [12,13], termina­tion of long-chain fatty acid synthesis [14], stimulation of choles­terol to the inner mitochondrial membrane [3,15]' inhibition of glucose-induced insulin release from pancreatic islets [16] , and stimulation of mitochondrial steroidogenesis [3,15,17].

Human DBI is an 86-amino acid polypeptide that has been puri­fied to homogeneity from human brain [18,19] and the human gene has been localized to chromosome 2, within the 2q12-21 region [20] . Non-neuronal DEI is highly concentrated in liver, kidney, and

Manuscript received February 12, 1993; accepted for publication June 21, 1993.

Reprint requests to: Dr. Hannu Alho, Department of Biomedical Sciences, University of Tampere, Box 607, 33101 Tampere, Finland.

Abbreviations: CBR, central benzodiazepine receptor; OBI, diazepam binding inhibitor; DBI-LI, diazepam binding inhibitor-like immunoreac­tivity; MBR, mitochondrial benzodiazepine receptor; NGS, normal goat serum; PAP, peroxidase-anti peroxidase. .

dist.ributed throu~hou.t t~e cytoplasm. Although the physio­logIc role of OBI m skm IS unknown, our results indicate that OBI may serve as an endogenous ligand for mitochondrial benzodiazepine receptors. Its activity could be related to the regulation of lipid and cholesterol synt!lesis in keratinocytes and sebaceous glands and to the secretIon of sweat in sweat glands. Key words: diazepam binding inhibitor/benzodiaze_ pine receptors/immunocytochemistry/human skin/lipid synthesis.] Invest Dermato/101 :800-803, 1993

endocrine tissues [21]. but there is no previous report on the locali­zation or distribution of DB I in skin. Recent observations that MER is also localized in skin [6] and that DEI stimulates steroid synthesis via MER .could be o~ special i~terest in studies of the physiologic and pathologiC metabolism of sk1l1. These recent findings prompted us to investigate the distribution and localization of DEI-like immu­noreactivity (DBI-LI) in human skin.

MATERIALS AND METHODS

Tissue Preparation Skin samples were obtained from three male and four female. patients ~ged 18 - 82 years. The Ratients were operated on for benign or mahgnant s~m tumors or for cosmetic reasons. Only healthy skin pieces were mcluded m the present study. The samples were taken from the skin of face, neck, upper arm, back, or abdomen and immersed in 4% para formal de­hyde containing 0.3% picric acid in 0.1 M phosphate buffer, pH 7.3, for 2-4 h at 4 · C. Before the immunohistochemical staining, the specimens were washed in phosphate-buffered saline (PBS) for 48 h, embedded in OCT-Tissue-Tek II (Miles Inc., Elkhart,IN), and cuton a cryostat at 10 /lIn. The sections were stored at -70 · C until use.

P.rin;ta~ ~tibody A polyclonal antiserum against human diazepam bmdmg mhlbltor (OBI) was used. The antIserum was raised in rabbits against human OBI, extracted, and purified from human postmortem brain. The characterization of h-DBI antiserum is described in detail by Ferrem et al [11].

Immunohistochemistry For the demonstration of OBI immunoreactiv_ ity the sections were incubated with h-DBI antiserum (1: 1000) in PBS containing 1% IGS for 24 h. The staining procedure has been previously described in detail by Alho et at [22,23] . The sections were mounted Ut Aquamount (Gurr, BDH, Dorset, U.K.) and observed with a Nikon Micro­phot-FXA light microscope. The immunohistochemical specificity Was verified by incubating the sections either with preimmune serum instead of the specific antiserum or by preabsorbing the OBI antiserum with OBI peptide (1.5 11M).

Electron Microscopy After the tissue preparation and immunohisto­chemical staining procedure described above, a conventional electron mi. croscopy method was employed. Briefly, the sections were postfixed ",;th 2.5% glutaraldehyde in 0.1 M PBS for 1 h at room temperature, posts tained with ice-cold 2% osmium tetroxide in 0.1 M PBS for 1 h, dehydrated in

0022-202X/93/S06.00 Copyright © 1993 by The Society for Investigative Dermatology, Inc.

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VOL. 101, NO. 6 DECEMBER 1993

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b

c -

DBI IMMUNOREACTIVITY IN HUMAN SKlN 801

Figure 1. DDI-LI in the human epidermis and sebaceous gland as demon­strated with the immunoperoxidase method. a) In epidermis an intense staining is seen in the granular cell layer (arrowhead) and in the upper parts of the spinous cell layer but not in the basal cell layer (asterisks) or keratin layer (opell arrow). b) A control section incubated with preabsorbed DBI anti­serum; no specific staining is seen. c) In sebaceous gland the most intense staining is seen in the outer sebaceous cells (arrowheads). Bar, 50 jim .

ethanol, and, via propylene oxide, embedded with Epon and cut into ul­trathin sections and counterstained with lead citrate only (0.2%).

RESULTS

The distribution ofDBI immunoreactivity (DBI-LI) was similar in all the tissue samples studied. No differences were noted in the staining intensity or distribution of DBI-LI between males and fe­males or between different skin regions. In the epidermis keratino­cytes in the upper part of the spinous layer and throughout the granular cell layer showed DBI-LI (Fig 1a) The intensity of the immunoreaction increased in the more superficial cell layers. How­ever, no DBI-LI was seen in the corneal layer (Fig 1a). The immu­noreaction was seen in the cytoplasm of the keratinocytes, and the nuclei were unstained. Intense DBI-LI was also seen in the eccrine sweat glands. Cytoplasmic staining was seen in the secondary coils of the glands but not in the ductal parts of the glands (Fig 20,b). DBI-LI was also demonstrated in the sebaceous glands. Immunore­activiry was seen in the cytoplasm of sebaceous cells, whereas the lipid droplets as well as the cell nuclei were unstained (Fig lc). None of the other dermal structures showed any DBI-LI.

No DBI-LI could be demonstrated in the control sections incu­bated with primary antibody preabsorbed with DBI peptide (Fig lb) or with the preimmune serum.

In the eccrine sweat gland DBI-LI was localized, using the elec­tron microscopy (EM) technique, in the clear cells of the secretory coil. No immunostainillg was observed in the dark cells of secretory coil or in any other cells of the ductal parts of the glands (Fig 30) . DBI was distributed throughout the cytoplasm. Precipitates were seen in Golgi apparatus and mitochondria. A similar distribution of DBI-LI throughout the cytoplasm was observed in the keratino­cytes and in the secretory cells of sebaceous glands (Fig 3b).

DISCUSSION

This study is the £lIst demonstration ofDBI-LI, a putative endoge­nous modulator of mitochondrial benzodiazepine receptors and fatty acyl-CoA binding protein, in human skin. In normal human skin we demonstrated the presence ofDBI-LI in the clear cells of the eccrine sweat gland, secretory cells of the sebaceous gland, and in granular cells of the epidermis. The peptide was undetectable in other cell types of the skin.

The epidermal location of DBI-LI correlates with the whole­body autoradiographic localization of the mitochondrial-type ben­zodiazepine receptors in neonatal rat skin, as demonstrated by An­holt et 01 [6]' With theLl technique it was not possible to establish the localization of the receptors to any distinct epidermal layer or dermal adnexes. Further electron microscopic evidence is needed to determine whether the intracytoplasmic DBI-LI demonstrated here is co-localized w ithin the same cells with MER. However, it has been shown that many cells containing DBI are also highly enriched in MERs [21) .

Although DB! is widely distributed in various peripheral tissues, it is expressed in each tissue in specialized cells. DB! is highly concentrated in steroid producing cells, such as Leydig cells in testis and cortical cells of the 3drenal gland [21,25), as well as in cells regulating water and electrolyte transport, such as distal convoluted duct cells in the kidney [21]. Although the activity of DBI in pe­ripheral tissues is still far fr0111 clear, there is evidence to show that it

802 ALHO ET AL

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Figure 2. Distribution of DBI-Ll in the eccrine sweat gland. a) The secre­tory coils of the glands ate immunoreactive (asterisks) whereas the ductal parts (circles) are unstained. b) A serial section counterstained with hematox­ylin-eosin. Bar, 50 iJ.m.

is involved in steroid and cholesterol biosynthesis, water and elec­trolyte transport, acyl-eoA binding, and glucose-mediated insulin release [3,13,16]. In the light of these possible functions, our immu­nocytochemical findings are of special interest.

The cells of sebaceous glands exhibited heterogeneous immuno­reactivity probably corresponding to the narrow cytoplasmic rims containing the cell organelles and the lipid synthesis. In the epider­mis, DBI-LI was restricted to the uppermost layers ofkeratinocytes. EspeciaIly the cells of the granular layer were immunoreactive. These cells are known to synthesize, contain, and excrete lipids with a remarkably high proportion of cholesterol in epidermal lipids [28]. One important component for lipid and steroid biosynthesis is fatty acyl-CoA (12] and it is possible that DBI via binding acyl-CoA stimulates both cholesterol and lipid synthesis.

Recently DBI-LI has also been localized in the epithelial cells of various rat organs that are specialized in water and electrolyte reab­sorption and secretion, such as epithelial cells of kidney tubules, upper intestinal tract, bronchioles, ana choroid plexus and cLIcum­ventricular organ in the brain (21,25] . Our results demonstrate that in the sweat gland, DBI-LI was localized in the clear cells of the gland, which are mainly responsible for the secretion of sweat [26,27].

Our findings suggest that DBl might also be metabolically in­volved in the synthesizing and secretion process of lipids in kerati-110cytes. The forming and composition of an epidermal lipid barrier

THE JOURNAL OF INVESTlGATIVE DERMATOLOGY

Figure 3. Expression of DBI-LI in human skin at the ultrastrucrurallevcl (pre-embedding PAP technique). a) Sweat gland. DBI-immunoactiviry is seen in a clear cell while a neighboring cell is negative (asterisks) ,. the immu­noreactivity is distributed throughout the cell cytoplasm including mit()­chondria (m) . The nucleus (11) is unstained; bar, 111m. b) Sebaceous gland. DBI-LI is distributed throughout the narrow cytoplasmic rim of the cell including the mitochondria (m). The sebum (asterisks) is unstained. Bar, 500 nm.

as weIJ as the composition of the sebum in the sebaceous glands could be modified by the activity of DBl. The demonstration of OBI in skin has a special interest because OBI may have multiple important functions in skin physiology and pathologic processes in metabolically different types of skin disease.

Tit is work was supported by thc S.jusilius Foundatiott attd tlte Acadcmy ofFitt/and.

REFERENCES

1. Wang JKT, Morgan JI, Spector S: Ben7.odiazcpincs that bind at peripheral sitts inhibit cell proliferation. Proc Nail Acad Sci USA 81 :753-756, 1984

2. Ruff MR, Pert CB, W eber RJ, el al: Benzodiazepine receptor-mediated chemcr taxis of human monocytes. Sci ... c" 29: 1281- 1283, 1985

3. Desman MJ, Yanagibashi K, Lee TD, Kawamura M, HalJ PF, Shively JE: Identifi­cation of des-(Gly-I1c)-endozepine as an effector of corticotropin-depend~.nt adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by th, peripheral benzodiazepine receptor. Prot Noel Acad Sci USA 86:4897 -4901. 1989

4. Krueger ICE: Peripheral-type benzodiazepille receptors: a second site of action fu< benzodiazepilles. Neuropsl'chopharmocologl' 4:237 - 244, 1991

5. Anholt MH, Pendersen PL, Dc Sousa EB, Snyder SH: The peripheral-type ben­zodiazepine receptor: location to mitochondria! outer membrane. J Bioi Chm. 261:576-583,1986

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6. Anholt R, de Sousa E, Oster-Granite ML, Snyder S: Peripheral-type benzodiaze­pine reseptors: autoradiographic localization in wholebody sections of neonatal rats.] Pharmacol Exp Ther 233:517 - 526, 1985

7. Mukhin AG, Papadopoulos V, Costa E, Krueger KE: Mitochondrial benzodiaze­pine reseptors regulate steroid biosynthesis. Proc Nad Acad Sci USA 86:9813-1916,1989

8. Costa E: "Prolegomena" to the biology of the diazepam binding inhibitor (OBI). Neuropharmacology (30(12B):1357 - 1364, 1991

9. Costa E, Guidotti A: Diazepam binding inhibitor (OBI): a peptide with multiple biological actions. Life Sciences 49:325 - 344, 1991

10. Guidotti A, Forchetti CM, Corda MG, Konkel 0, Bennett DC, Cosra E: Isola­tion, characterization and purification to homogeneity of an endogenous poly­peptide with agonistic action on benzodiazepine receptors. Proc Natl Acad Sci USA 80:3531-3533,1983

11. Ferrero P, Santi MR, Conti-Tronconi B, CostaE, Guidotti A: Study of an octade­caneuropeptide derived from diazepam binding inhibitor (OBI): biological activity and presence in rat brain. Proc Nad Acad Sci USA 83:827 -831. 1986

12. Knudsen J: Acyl-CoA-binding protein (ACBP) and its relation to fatty acid-bind­ing protein (FABP): an overview. Mol Cell Bioci,em 98:217-223,1990

13. Knudsen J : Acyl-CoA-binding and transport an alternative function for diazepam binding inhibitor (OBI), which is identic.,1 with acyl-CoA-binding protein.Neuropharmacology 30:1405-1410,1991 .

14. Mikkelsen J, H0jrup P, Nielsen PF, Roepstorff P, Knudsen J: Amino aCId se­quence of acyl-CoA-binding protein from cow liver. Bioe/rem] 245:857 -861, 1987

15. Krueger KE: Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells. ] Bioi CI,em 265:15015-15022. 1990

16. Borboni P, Condorelli L. De Stefanis P, Sesti G, Lauro R: Modulation of insulin secretion by diazepam binding inhibitor and its processing products. Neurophar­macology 30:1399-1403, 1991

11. Papadopoulos V. Berk.ovich A. Krueger KE: The role of diazel'am binding inhibi­tor and Its processmg products at mltochondnaI benzodlazepme receptors: regulation of steroid biosynthesis. Neuropharmacology 30:1417 - 1423, 1991

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

OBI IMMUNOREACTIVITY IN HUMAN SKIN 803

Ferrero p. Costa E. Conti-Tronconi B. Guidotti A: A diazepam binding inhibitor (DBI)-like neuropeptide is detected in human brain. Brai" Res 399:136- 142. 1986

Shoy. b M. Gentry LE, Marquardt H , Todaro GJ: Isolation and characterization of a putative endogenous benzodiazepine (endozepine) fro m bovine and human brain.] Bioi Chelll 261:11968-11973, 1986

DeBernardi M. Crowe R, Mocchetti I. Shows T, Eddy R, Costa E: Chromosomal localization of the human diazepam binding inhibitor gene. Proc Nad Acad Sci USA 85:6561-6565, 1988

Bovolin P, SchlichtingJ, Miyata M, Ferrarcse C, Guidotti A, Alho H: Distribu­tion and characterization of diazepam binding inhibitor (OBI) in peripheral tissues of rat. Regulatory Peplides 29:267 -281, 1990

Alho H, Cost~ E~ Fe~rero P, Fujimoto M, Consenza-Murphy 0, Guidotti A: Dlazepam-bmd111g mhlbltor: a neuropeptide located in selected neuronal pop_ ularions of rat brain. SciCli" 229: 1 79 - 182, 1985

Alho H, Fremea~ RT, Ti,:,dge H, Wilcox J , Bovolin P, Brosius JL, Costa E: DIazepam b111dmg mhlbltor gene expression: location in brain and peripheral tissues of rat. Proc Natl Acad Sci USA 85:7018-7022, 1988

Rheaume E, Tonon Me, Smith F, Simard J , Desy L, Vandty H, Pelletier G: Location of the endogenous benzodiazepine ligand octadeeaneuropeptide in the rat testes. Elldocrillology 127:1986- 1994, 1990

Alho H, Harjuntausta T , Schultz R, Pelto-Huikko M. Bovolin P: Immunohisto­chemistry of diazepam binding inhibitor (OBI) in the central nervous system and penpheral organs: Its possible role as an endogenous regulator of different types of benzodiazepine receptors. Neuropharmacology 30(12B):1381 - 1386. 1991

Sato K: The physiology and pharmacology of the ecctine sweat gland. In: Gold­slDlth LA (cd.). Biochemislry alld PhYSiology of the Skill, Vol. 1. Oxford University Press, New York, Oxford, 1983. pp 596-641

Sato K, Kang W , Sam KT: Biology of sweat glands and their disorders. I. Normal sweat gland function.] Am Acad Dermalol20( 4):537 - 563, 1989

Yardley HJ: Epidermal lipids. In: Goldsmith LA (ed.). Biochemistry alld Physiology of lire Ski,l. Vol. 1. Oxford University Press, New York, Oxford. 1983. pp 363-381