Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 254–258

Expression of steroidogenic factors 1 and 2 in normal human pancreas

Angelica Morales a,∗, Felipe Vilchis a, Bertha Chavez a, Carlos Chan b,Guillermo Robles-Dıaz c, Vicente Dıaz-Sanchez d

a Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran,Vasco de Quiroga #15 Tlalpan, 14000 Mexico DF, Mexico

b Department of Surgery, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran,Vasco de Quiroga #15 Tlalpan, 14000 Mexico DF, Mexico

c Department of Experimental Medicine, Faculty of Medicine, Universidad Nacional Autonoma de Mexico, Mexicod MEXFAM A.C. Juarez, 208 Tlalpan, 14000 Mexico DF, Mexico

Received 25 July 2005; accepted 25 October 2005

Abstract

Orphan nuclear receptor steroidogenic factor-1 (SF-1) is crucial for development and function of steroidogenic organs. The steroidogenicfaofIdwds©

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actor-2 (SF-2) is an essential factor involved in cholesterol transfer and activation of promoters of steroidogenic enzymes CYP11A1, CYP17nd Steroidogenic Acute Regulatory Protein (StAR). We have previously demonstrated steroidogenic activity in pancreatic tissue. The aimf this study was to investigate the presence of SF-1 and SF-2 in human pancreas. Total RNA was extracted from normal male (five) andemale (five) samples, obtained from the organs donor program. RT-PCR approach was used to analyze the expression of SF-1 and SF-2.mmunohistochemical analysis was performed for SF-1. The bands of expression were present in both male and female samples, althoughifferential expression was observed. For both factors, the signal detected was more evident in males than in females. A similar patternas present in the immunohistochemical study. Normal human pancreas expresses SF-1 and SF-2 factors similarly to ovary and adrenals. Aistinctive characteristic is the sexually dimorphic expression of these factors. Our data provide evidence suggesting that the pancreas achievesteroidogenic activity supporting the presence of gender- and location-related differences in the expression of these steroidogenic factors.

2006 Elsevier Ltd. All rights reserved.

eywords: Normal human pancreas; Orphan nuclear receptors; Steroidogenic factor-1 (SF-1); Steroidogenic factor-2 (SF-2); Steroidogenesis; NR5A1; NR5A2

. Introduction

Cytochrome P450 (CYP) and hydroxysteroid dehydroge-ase enzymes are involved in the conversion of cholesterolo steroid hormones. These enzymes are primarily expressedn gonads, adrenal glands and placenta [1,2]. Interestinglyome of these enzyme activities have also been demon-trated in non-steroidogenic endocrine tissue such as brain,eart and pancreas [3–5], where they may be involved inmportant paracrine and autocrine actions. Recently defineduclear receptor subfamily NR5A includes three highlyelated orphan receptors, so named for their lack of knownigands. One member of this subfamily is steroidogenic

∗ Corresponding author. Fax: +52 55 54 87 00 42.E-mail address: Angelica170969@aol.com (A. Morales).

factor-1 (SF-1), also known as adrenal 4-binding protein(Ad4BP) encoded by the NR5A1 gene [6]. This factor is amammalian homologue of Drosophila’s fushi tarazu factor(Ftz-F1) and structurally belongs to a subgroup of receptorsthat includes those for steroids, thyroid and retinoid hor-mones. Transfection studies using different cellular lines havehighlighted important roles for SF-1 in the transcriptionalactivation of elements present in the promoters of variousgenes that encodes steroidogenic enzymes. Embryologicalexpression studies and targeted gene disruption analyseshave demonstrated a critical role for SF-1 in the devel-opment and function of the primary steroidogenic organssuch as the adrenal glands and the ovary. Moreover, it hasbeen observed that the SF-1 knockout mice lack of adrenalglands and usually die within the first week of postnal lifebecause of adrenocortical insufficiency [7–11]. They also

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A. Morales et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 254–258 255

present impairment in expression of pituitary gonadotropinsas well as agenesis of the ventromedial hypothalamic nucleus(VMH), confirming roles of SF-1 at all three levels of thehypothalamic–pituitary–steroidogenic organ axis [12].

Another member of the NR5A orphan nuclear receptorsubfamily is steroidogenic factor-2 (SF-2), also known ashB1F/Hlrh-1, LRH-1, FTF or CPF encoded by the NR5A2gene [13]. This factor is involved in a wide range of bio-logical processes, it has been recognized in cholesteroltransfer metabolism as well as in activation of promotersof steroidogenic enzymes including P450scc (CYP11A1),P450c17 (CYP17) and by the steroidogenic acute regula-tory (StAR) protein [14]. In this study, we sought whetherthe human pancreas shared the expression of steroidogenicfactors involved in the regulation of steroid hormones produc-tion. Here, the expression and localization of two member ofthe NR5A nuclear receptor subfamily in the normal humanpancreas are reported.

2. Materials and methods

2.1. Preparation of RNA

The organs donor program kindly provided 10 human pan-creas samples, 5 males and 5 females. The samples (10 ge−fiTuelf

2

tDfutecwaCTSnGTwt

for 2 min; 30 cycles (94 ◦C, 45 s; 65 ◦C, 45 s; 72 ◦C, 45 s);followed by a 3 min final extension at 72 ◦C. RNAs withoutRT were used as negative control. The number of cycles wasselected to be in the linear part of the amplification curve. Fivemicroliters from each reaction was analyzed on 1% agarosegels containing ethidium bromide and evaluated by densitom-etry using the Eagle Eye II still video system (Stratagene, LaJolla, CA). Quantification depicts relative changes betweentranscripts of males and females.

2.3. Nucleotide sequencing

Sequencing analyses of the RT-PCR fragments wereperformed in both directions using the thermosequenase[�-33P]ddNTP terminator cycle sequencing kit (Amer-sham, Cleveland, OH), and the oligonucleotide primersabove described. Sequencing reaction was run on 6%polyacrylamide–7.5 M urea gels.

2.4. Immunohistochemical procedure

A fragment of each pancreatic tissue was fixed in 4%paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for3–6 h. Sectioning at 7 �m was performed according to stan-dard procedures. Sections were deparaffinized and thenendogenous peroxidase activity was eliminated from the sec-t4pwrwbS(pfog

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aFfTSptfwc

ach) were frozen on liquid nitrogen and then stored at70 ◦C until analysis. Samples of each specimen were used

or RNA preparation and for immunohistochemical stud-es. Total RNA was isolated from pancreatic tissue, usingRIZOL reagent (Life Technologies, Inc.), according to man-facturer’s instructions. The amount and quality of RNA werestimated by spectrophotometer at 260–280 nm. Due to theow quantity recovered in each individual sample, male andemale samples were pooled according to gender.

.2. RT-PCR analysis

Total RNA was subjected to reverse transcription withhe first strand cDNA synthesis kit for RT-PCR (Rocheiagnostics, Mannheim, Germany) following the manu-

acturer’s instructions. Complementary DNA was preparedsing 1.0 �g RNA and 20 U of AMV reverse transcrip-ase. From the 20 �l RT reactions, 2.0 �l was then used inach 25 �l PCR primed with gene specific primers. Oligonu-leotides used for analysis of SF-1 transcripts by RT-PCRere derived from the GenBank accession no. NM004959

nd are: SF33, 5′-CAC TGG CTG GCT ACC TCT ACC CTG-3′; SF4, 5′-GCC TTC TCC TGA GCG TCT TTC ACC-3′.he forward and reverse primers used for amplification ofF-2 transcripts were derived from the GenBank accessiono. NM003822 and are: SF2F, 5′-TAT GCC CTC TGA CCTAC CAT TTC C-3′; SF2R, 5′-GAC TTG TTC CTG GAC ACCTC TAC C-3′. A set of oligonucleotides for cyclophilin [15]hich yielded a product of 453 pb was used in parallel reac-

ions as control. The PCR program was as follows: 94 ◦C

ions by incubation with 3% H2O2 in absolute methanol for0 min at room temperature and washing twice in PBS buffer,H 7.4. After blocking of non-specific binding componentsith 5% normal goat serum and 1% BSA in PBS for 1 h at

oom temperature, the primary antibody (anti SF-1, 1:150)as overlaid upon each section for 12 h in a humid cham-er at 4 ◦C (Santa Cruz Biotechnology, cat. no. sc-10976).ections were incubated with a rabbit anti-goat IgG-HRP1:100) used as a second antibody for 60 min at room tem-erature. Immunoreactivity was detected with DAB substrateor 10 min. The specificity of immunolabeling was tested bymitting the primary antibody. Sections of human adrenalland and ovary were used as positive controls.

. Results

.1. RT-PCR of SF-1/NR5A1 and SF-2/NR5A2

The expression of mRNA for SF-1 and SF-2 in pancre-tic tissue was demonstrated by RT-PCR. As can be seen inig. 1, PCR products of the expected sizes were amplifiedrom cDNA derived from male and female pooled samples.he relative abundance of specific transcripts for SF-1 andF-2 was consistently higher in male pancreas than in femaleancreas. DNA transcripts generated by PCR were sequencedo confirm identity. The nucleotide sequence of the 825-bpragment (SF-2, nt 675–1500) from male and female pancreasas found to be identical to the sequence of human NR5A2

DNA clone (accession no. NM003822); similar results were

256 A. Morales et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 254–258

Fig. 1. Representative expression pattern of SF-1 and SF-2 mRNA in humanpancreas. One microgram of total RNA each from male pancreas (lanes 1and 3) and female pancreas (lanes 2 and 4) was subjected to RT-PCR forSF-1, SF-2 and also cyclophilin (Cy). PCR products of 650 bp (SF-1), 825 bp(SF-2) and 450 bp (Cy) were revealed by 1.0% agarose gel electrophoresis.MW, 100-bp DNA ladder.

obtained when the 654-bp fragment (SF-1, nt 723–1377) frommale pancreas was sequenced (data not shown).

3.2. Immunohistochemistry

Immunohistochemistry was used to investigate the expres-sion of steroidogenic factor SF-1 in different regions of thenormal human pancreas. The analysis was performed usingsections of pancreas and sections of adrenal gland and ovaryas controls. Positive staining for SF-1 was obtained in bothmale and female pancreatic tissues. The labeling was specificof exocrine cells. In female samples, the immunoreactivitywas localized mainly in the cytoplasm of interstitial cells(Fig. 2), whereas in male tissue the label was found basi-cally in cytoplasm of ductal cells (Fig. 3). In both sexes therewas also labeling in the endothelium of blood vessels. Noimmunolabeling was found in the negative controls of thepancreas which were stained in the absence of the primaryantibody.

Fig. 2. Representative immunohistochemical staining for steroidogenic factor 1 inthe cytoplasm of interstitial cells (panels 1 and 2), and the endothelium of blood vesimmunolabeling was found in pancreatic islets (panel 5). Non-immune control sect

female normal human pancreas. Positive immunoresponse was localized insels (panel 3). Non-immune control sections of blood vessels (panel 4). Noion of pancreatic islets (panel 6).

A. Morales et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 254–258 257

Fig. 3. Immunohistochemical staining for steroidogenic factor 1 in male normal pancreatic tissue. Positive label was found mainly in ductal cells (panels 1 and2), and the endothelium of blood vessels (panel 3). No immunolabeling was found in pancreatic islets (panel 4).

4. Discussion

SF-1 in combination with other factors governs a sub-stantial number of genes that are involved in tissue func-tion and differentiation [16]. In addition to the gonad, SF-1transcripts have been detected in adrenal, pituitary, hypotha-lamus, spleen and placenta. Gene disruption studies haveclearly indicated that SF-1 plays key roles in the differen-tiation process of both non-steroidogenic and steroidogenictissues [17]. In this study, we present evidence of the expres-sion of SF-1 and SF-2 in normal adult pancreatic tissue, aswell as the cell types that express specific immunolabeling.This report expands the research line of our group in the char-acterization of the pancreas as a steroidogenic tissue [18–20].The results presented here demonstrate that in male normalpancreatic tissue SF-1 and SF-2 messages are present, andin higher amounts than those observed in the female normalpancreas. This type of sexually dimorphic pattern of expres-sion has been reported for SOX 9, GATA-4 and SF-1, duringembryonic male gonadal development. In fact, there are datathat suggest that SOX 9 up-regulates SF-1 and accounts par-tially for the sexually dimorphic expression pattern of SF-1observed during male gonadal differentiation [21]. In new-born rat, the expression of both SF-1 and SOX 9 diminishesand is concomitantly up-regulated before puberty [22].

Whether the same pattern continues during adulthood,itfc

located in the cytoplasm of interstitial cells, whereas in themale samples the label was observed mainly in the ductalcells. These differences might be of importance in clinicalcases, because adult ductal cells share some similarities withembryonic primitive ducts and based on challenged pancreasregeneration experiments, the adult ductal cells have beenproposed to be pancreatic stem cells [23]. In addition, inthe main form of pancreatic cancer, pancreas adenocarci-noma that is predominant in male subjects, tumor cells sharesimilarities with ductal cells [24–26]. The presence of spe-cific immunolabeling for SF-1 in the endothelial cells of theblood vessels in both male and female pancreas might berelated to the vascularization of the tissue. Expression ofthe gene encoding SF-1 has been confirmed in endothelialcells of the splenic sinus and pulp vein [27], and furthermore,SF-1 ablation induces defects in the establishment of splac-nic vascularization as well as defects in erythropoiesis [28].The presence of SF-1 in the endothelial cells of the bloodvessels might be related to the novel family of angiogenicmitogens recently characterized. Endocrine gland derivedvascular endothelial growth factor (EG–VEGF) is a highlyrelated endothelial cell mitogen and chemotactic factor withrestricted expression profiles and selective endothelial cellactivity. The expression of human EG–VEGF occurs predom-inantly in steroidogenic glands, and consistent with such anexpression pattern, the human EG–VEGF gene promoter hasa

op

n the gonads or another steroidogenic or non-steroidogenicissue, remains to be demonstrated. Another dimorphic dif-erence between male and female pancreas is the type ofells expressing SF-1. In females the label was predominantly

potential binding site for SF-1 [29].The presence of SF-1 transcripts in the endothelial cells

f the blood vessels and the steroidogenic properties of theancreas are two characteristics that might account for the

258 A. Morales et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 254–258

possibility that EG–VEGF could be the regulatory factor inangiogenesis in this tissue. It is very well known that angio-genesis is a process related to tumor growth and malignancy.Recent studies have demonstrated that vascular endothelialgrowth factor expression correlates with microvessel densityand tumor progression [30]. The identification of the selec-tive angiogenic molecules in pancreas may be of relevancefor devising therapeutic strategies in pancreatic tumors. Thedata presented herein provide more elements for the under-standing of the complex pancreatic physiology and point outthe sexually dimorphic nature of this organ.

Acknowledgments

We thank Veronica Rodriguez for her invaluable technicalassistance, and Dr. Andres Castell for the histological evalu-ation.

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