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Endocrinology Vol. 140, No. 6 2771-2780
Copyright © 1999 by The Endocrine Society

ARTICLES

Bovine Thrombospondin-2: Complete Complementary Deoxyribonucleic Acid Sequence and Immunolocalization in the External Zones of the Adrenal Cortex1

Marc Danik2,3, Anna M. Chinn2, Bruno Lafeuillade4, Michelle Keramidas, Stéphanie Aguesse-Germon5, Armelle Penhoat, Hui Chen6, Deane F. Mosher, Edmond M. Chambaz and Jean-Jacques Feige

Institut National de la Santé et de la Recherche Médicale U-244, Laboratoire de Biochimie des Régulations Cellulaires Endocrines, Département de Biologie Moléculaire et Structurale, CEA/Grenoble (M.D., A.M.C., B.L., M.K., S.A.-G., E.M.C., J.-J.F.), F-38054 Grenoble Cedex 9, France; Institut National de la Santé et de la Recherche Médicale-Institut National de la Recherche Agronomique U-418, Communications Cellulaires et Différenciation, Hôpital Debrousse (A.P.), 69322 Lyon Cedex 5, France; and the Department of Medicine, University of Wisconsin (H.C., D.F.M.), Madison, Wisconsin 53706

Address all correspondence and requests for reprints to: Dr. Jean-Jacques Feige, INSERM U-244, Laboratoire de Biochimie des Régulations Cellulaires Endocrines, Département de Biologie Moléculaire et Structurale, Commissariat à l’Energie Atomique/Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France. E-mail: jjfeige{at}geant.ceng.cea.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAMMALIAN adrenal cortex is a dynamic organ that undergoes rapid enlargement during the latter two thirds of intrauterine life, important degeneration/regeneration processes during the early postnatal period, and constant renewal during adult life (1, 2). These processes are under the main control of the hypothalamo-pituitary axis. The pituitary hormone ACTH is clearly the major trophic factor for adrenocortical cells both in vitro and in vivo.

Given the variety of biological functions controlled by ACTH in the adrenal cortex, ranging from the stimulation of steroid biosynthesis to the control of trophicity, cell shape, cell survival, etc., we hypothesized that some extracellular proteins act as biological relays for ACTH. Such proteins would be synthesized and secreted under the control of ACTH and be implicated in specific functions triggered by the pituitary hormone. Biochemical analysis of the proteins secreted by primary cultures of bovine fasciculata-reticularis cells led us to the characterization of a large trimeric protein that we named corticotropin-induced secreted protein (CISP) because its concentration in the culture medium increased 5- to 14-fold (depending on cell preparations) in the presence of ACTH (3). CISP was purified to apparent homogeneity from the conditioned medium of ACTH-treated bovine adrenocortical (BAC) cells of fasciculata-reticularis origin. Amino acid sequencing of its N-terminus and of three derived tryptic peptides revealed strong homology with the sequences of mouse and human thrombospondin-2 (TSP2) (3).

TSPs constitute a family of extracellular proteins comprising five distinct members that share sequence homology and a multimodular structural organization (4, 5). TSP1 and TSP2 both are homotrimeric. Their subunits contain an N-terminal heparin-binding domain, a procollagen homology domain, three type I repeats, three type II repeats, seven type III repeats (calcium-binding sites), and a C-terminal globular domain. TSP3, TSP4, and COMP/TSP5 are pentameric, and their subunits lack the procollagen and type I domains present in TSP1 and TSP2. Sequence comparison of TSP1 and TSP2 shows a gradient of increasing identity extending from the N-terminus toward the C-terminus. TSPs possess multiple types of cell surface receptors that recognize discrete domains of the molecule (6). These include heparan sulfate proteoglycans (7), low density lipoprotein receptor-related protein (8), CD36 (9), CD47 (10), and integrins. Thus, it is not surprising that TSPs are implicated in a number of biological functions, including cell adhesion, cell spreading, cell migration, cell proliferation, angiogenesis, and neurogenesis (11, 12).

The aim of the present study was to clone and sequence the CISP complementary DNA (cDNA) to determine whether the previously described CISP sequence represented the bovine ortholog of TSP2 or another TSP family member only partially related to TSP2. Moreover, the size of purified CISP monomers (195 kDa) appeared larger than the reported size of recombinant mouse TSP2 monomers (180 kDa). Whether this difference resulted from differential proteolytic cleavage or differential glycosylation of the two proteins, or whether it indicated that the CISP gene differed from the TSP2 gene by the presence of an additional structural module was not clear before this study. Presented here are the results from the cloning of overlapping cDNAs encoding full-length CISP as well as those from comparative TSP2 deglycosylation experiments. These results establish that CISP is the bovine ortholog of TSP2. This study also reports the distribution of TSP2 in various bovine organs as well as its immunolocalization in the bovine adrenal gland.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Restriction enzymes were obtained from Eurogentec (Seraing, Belgium). [{alpha}-32P]Deoxy (d)-CTP (>800 Ci/mmol) and [{alpha}-35S]dATP (>1000 Ci/mmol) were purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Calf intestinal phosphatase and T4 DNA ligase were obtained from Boehringer Mannheim (Meylan, France), and recombinant peptide N-glycosidase F was purchased from Genzyme Corp. (Cambridge, MA). Ampicillin and tetracycline were obtained from Sigma Chemical Co. (St. Louis, MO). Heparin-agarose and Sephacryl S-400 were purchased from Pharmacia Biotech (Uppsala, Sweden). The bovine 18S cDNA probe was synthesized by RT-PCR in our laboratory. Bovine CISP was purified from the conditioned medium of ACTH-treated (10-8 M; 24 h) BAC cells as previously described (3). Recombinant mouse TSP2 was produced in Sf9 insect cells after infection with the recombined baculovirus mTSP2 183/pEV/35K (13) and was purified by heparin-agarose chromatography. Polyclonal rabbit anti-mTSP2 antibodies were prepared by Eurogentec using baculovirus-expressed mouse TSP2 as the antigen. Polyclonal rabbit anti-CISP antibodies were developed in our laboratory as previously described (14). Immune and nonimmune IgGs were purified from antisera and normal serum, respectively, by Hi-Trap protein G (Pharmacia Biotech) chromatography. Biotinylated horseradish peroxidase complex was obtained from Amersham (Little Chalfont, UK). Standard molecular biological techniques were performed as described by Sambrook et al. (15).

BAC cell cDNA library construction
A BAC cell cDNA library was constructed in the pCDM8 vector (Invitrogen, La Jolla, CA) using polyadenylated [poly(A)+] RNAs purified from ACTH-treated BAC cells. Briefly, 108 bovine fasciculata-reticularis cells were cultured in serum-free medium (Ham’s F-12-DMEM, 1:1 mixture; Life Technologies, Cergy Pontoise, France) containing 5 µg/ml insulin, 10 µg/ml transferrin, and 10-4 M vitamin C. On day 3, cells were treated with 10-10 M ACTH for 48 h before extraction of total RNA and isolation of poly(A)+ RNA. Double stranded cDNA was synthesized from 3.75 µg poly(A)+ RNA using the First Strand cDNA synthesis kit (Pharmacia Biotech). After the addition of BstXI adapters, the cDNA was inserted into the BstXI site of pCDM8 and introduced into Escherichia coli strain MC1061/P3. One fifth of the transformants was amplified once, divided into aliquots, and stored at -80 C.

Library screening
Approximately 160,000 recombinant bacterial colonies from the amplified stock were plated and transferred onto duplicate nylon membranes (ICN Pharmaceuticals, Inc.) according to the manufacturer’s instructions. Screening of replicate colony lifts for CISP cDNA was performed using a 386-bp random primed RT-PCR product (16). Hybridization was performed for 63 h. Small areas (total of 23) containing positive colonies were separated into 4 pools. Each pool was plated and probed as described above, except that the hybridization time was reduced to 26 h. After an additional round of hybridization at low bacterial density, individual positive colonies were picked and regrown into agar stabs. Twenty of these colonies were analyzed on an agarose gel for plasmid size, and the presence of a CISP-related insert was confirmed by PCR analysis. The cDNAs carried by the two largest recombinant plasmids, p249c and p268c, were sequenced at their 3'-ends. Partial restriction mapping and sequencing suggested that these 2 clones were identical. The longest fragment containing CISP cDNA sequences (p268c) was subcloned as a 2.1-kb XhoI-BamHI fragment into pUC18 at the SalI and BamHI sites, generating clone p268c1. For sequencing, p268c1 was subcloned by PstI digestion and unidirectional deletion.

Rapid amplification of cDNA ends (RACE)-PCR
Two partial cDNAs, one encoding the C-terminal 666 amino acids and 689 nucleotides (nt) of the 3'-untranslated region (3'-UTR) and another bearing 2 kb of the 3'-UTR were cloned by RACE-PCR using the Marathon cDNA amplification kit (CLONTECH Laboratories, Inc., Montigny le Bretonneux, France). Briefly, on day 3 of culture, BAC cells were transferred to serum-free Ham’s F-12 medium for 24 h, then stimulated for 24 h with 10-8 M ACTH. Poly(A)+ RNA was isolated using the Poly ATtract 2000 system (Promega Corp., Charbonnieres, France). Double stranded cDNA was synthesized from 2 µg RNA and ligated to the Marathon cDNA adaptors. Two RACE-PCR reactions were performed according to the manufacturer’s instructions, using either the primer 5'-CTGCACAGTCACCTGTGCTGGAGGGATC-3' corresponding to nt 2009–2036 or the primer 5'-CAGTCCATCCCAGTGCTTCCAGC-3' corresponding to nt 4069–4091 of the bovine sequence and the AP1 primer included in the kit. A 2.7-kb band from the first reaction and a 2.0-kb band from the second reaction were isolated and cloned. Subsequent PCR reactions were performed as described in the text in 1.5 mM MgCl2, 0.2 mM dNTPs, and 0.3 µM each of the 4069–4091 primer and the primer 5'-CTGACAAGACAAGTCTTCTTCCTGAGC-3' corresponding to the reverse complement of nt 4341–4367 in the bovine sequence in 50 µl, with 2.5 U Taq polymerase added as a hot start. After 2.5 min at 94 C, 30 cycles of 1 min at 94 C, 1 min at 68 C, and 1 min at 72 C were performed, and the products were run on a 1.2% agarose gel. Templates were 5 ng pAC1 or pAC5 plasmid, 500 ng cow genomic DNA, and the equivalent of 5 ng polyadenylated RNA, either reverse transcribed or mock reverse transcribed.

Nucleotide sequence determination
Double stranded DNA was sequenced using the dideoxy chain termination method and a T7 sequencing kit (Pharmacia Biotech), a Sequenase DNA sequencing kit (U.S. Biochemical Corp., Cleveland, OH), or an automated sequencer (ABI Prism model 373, Genome Express S.A., Grenoble, France). In the latter case, both dye primer and dye terminator cycle sequencing kits were used. MacVector’s Assembly Lign software (Oxford Molecular Ltd., Oxford, UK) was used to piece together the sequences of overlapping DNA segments.

Western blot detection of bTSP2
Fresh bovine organs were obtained from the local slaughterhouse and homogenized with a Polytron (Brinkmann Instruments, Inc., Westbury, NY) in 2 vol buffer [20 mM Tris (pH 7.5) and 2 mM CaCl2] containing a cocktail of protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). After centrifugation at 30,000 x g for 30 min, the supernatants were filtered through gauze, and their protein content was determined using the microBCA kit (Pierce Chemical Co., Oud Beijerland, The Netherlands). One hundred micrograms of cytosolic proteins were separated by 8% SDS-PAGE under reducing conditions and electrophoretically transferred (60 V, overnight) onto a nitrocellulose membrane. After 1 h of incubation in blotting buffer (PBS, 0.1% Tween-20, and 5% powdered milk) to block nonspecific binding sites, the membrane was probed with polyclonal rabbit anti-CISP IgGs (20 µg/ml in blotting buffer) for 1 h at room temperature. The nitrocellulose was then washed extensively with blotting buffer, followed by a 1-h incubation with antirabbit IgG antibodies coupled to horseradish peroxidase (Bio-Rad Laboratories, Inc., Richmond, CA). The immunoreactive proteins were detected using the Renaissance chemiluminescence kit (DuPont NEN, Les Ulis, France).

Protein deglycosylation
The proteins to be analyzed were precipitated with 10% trichloroacetic acid; dissolved in 0.55 M Tris-HCl buffer (pH 8.6), 0.5% SDS, and 50 mM 2-mercaptoethanol; and denatured by heating at 100 C for 5 min. After cooling, the reaction mixture was diluted three times with water and adjusted to 1.25% Nonidet P-40, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonylfluoride. The digestion of N-linked oligosaccharide chains was carried out overnight at 37 C in the presence of 10 U/ml peptide N-glycosidase. Control samples did not receive the enzyme. Untreated and deglycosylated proteins were then separated by 6% SDS-PAGE.

Northern blot hybridization
Total RNA was extracted from slices of bovine adrenal zona fasciculata, zona glomerulosa, and adrenal medulla tissues and from primary cultures using the RNAgents kit (Promega Corp., Charbonnieres, France). Twenty micrograms of RNA from the tissue and primary cultures were loaded onto a 1% agarose-1.9% formaldehyde gel, electrophoresed, and vacuum-blotted onto a nylon membrane (Hybond-N, Amersham, Les Ulis, France), as described by Fourney et al. (17). The membrane was hybridized for 90 min at 65 C in RapidHybe Buffer (Amersham, Les Ulis, France) with a 32P-labeled, random primed, gel-purified PCR fragment comprising nt 4069–4367 of bTSP2. It was then washed for 20 min in 2 x SSC (standard saline citrate)-0.1% SDS at room temperature, for 10 min in 1 x SSC-0.1% SDS at 65 C, and for 10 min in 0.1 x SSC-0.1% SDS at 65 C. Hybrids were visualized and quantitated using a ß-imager (PhosphorImager, Molecular Dynamics, Inc., Sunnyvale, CA). The probes were then stripped by immersing the membrane twice in boiling 0.1% SDS and hybridized with a probe for the 18S ribosomal RNA overnight at 65 C in 5 x SSPE, 0.5% SDS, 5 x Denhardt’s solution, and 0.1 mg/ml salmon sperm DNA (sodium chloride, sodium phosphate, EDTA), then washed as described above.

Immunohistochemical localization of TSP2
Fresh bovine adrenal glands were obtained from the local slaughterhouse, sagittally cut into 5-mm thick slices, and immediately fixed in Bouin-Hollande solution. After overnight fixation, the slices were rinsed, dehydrated through graded ethanol, and embedded in paraffin. Eight-micron sections were deparaffinized and hydrated for standard indirect peroxidase immunohistochemistry. Briefly, the sections were microwaved for 5 min at 900 watts in 0.1 M citrate buffer, pH 6.0. They were then incubated for 1 h at room temperature in 3% donkey serum in 50 mM Tris-HCl buffer (pH 7.4), 0.9% NaCl, and 0.3% Tween-20. This step was followed by an overnight incubation at 4 C with 20 µg/ml purified anti-CISP IgGs. Endogenous peroxidases were blocked with 0.3% hydrogen peroxide for 30 min at room temperature. The sections were subsequently incubated for 45 min with biotinylated donkey antirabbit Ig (diluted 1:250), washed, and incubated for 30 min with horseradish peroxidase-labeled streptavidin (diluted 1:250). The chromogenic peroxidase reaction was performed using the metal-enhanced diaminobenzidine substrate kit (Pierce Chemical Co.). Sections were mounted in Merckoglass (Merck KGaA, Darmstadt, Germany). For controls, nonimmune IgGs were used instead of the primary antibodies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of CISP cDNA clones
CISP cDNA clones were isolated by screening a cDNA library generated from bovine adrenocortical fasciculata-reticularis cells treated for 48 h with 10-10 M ACTH. A 386-bp cDNA probe was previously obtained by RT-PCR using degenerate primers based on the N-terminal amino acid sequence of CISP and a TSP2 nt sequence conserved between species (16). It encoded the 128 N-terminal amino acids of the mature CISP protein and was used for screening. Hybridization to 160,000 recombinant bacterial colonies yielded 23 positive clones. The longest insert excised from these plasmids (p268c1) was 2066 bp long. It contained 500 bp of 5'-untranslated sequence and 1566 bp of coding sequence starting from an ATG translation initiation codon (Fig. 1AGo).



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  		Figure 1. Organization of bovine TSP2 cDNAs. A, Three overlapping cDNAs corresponding to both 4.7- and 6.0-kb transcripts were isolated by library screening (p268c1) or RACE-PCR (pAC1, pAC5). The coding sequence is shown by the hatched bar. Dotted lines show the presumed alignment of sequences in the 3'-UTR of each transcript. B, Details of the 3'-region of the two TSP2 transcripts shown in A. The positions of probes used for Northern blots (P1, P2, and P3) are shown. C, Primers in the 3'-UTR, shown as arrows in B, amplify a 1.2- or 0.3-kb product from different templates: 1 and 5, cDNA clones pAC1 and pAC5; G, bovine genomic DNA; +, reverse transcribed BAC cell RNA; -, mock reverse transcribed BAC cell RNA; S, Mr marker VI from Boehringer Mannheim.

 
An overlapping cDNA was generated by 3'-RACE-PCR using primers specific for the bovine TSP2 sequence. This 2.7-kb long clone (pAC1) encoded the C-terminal 666 amino acids plus 0.7 kb of 3'-untranslated sequence (Fig. 1AGo) and probably corresponds to the 3'-end of the shorter of the 2 transcripts visible on Northern blots of adrenocortical cell RNA (see Fig. 3Go and description below). Overlapping sequences were assembled into a 4697-bp cDNA comprising 500 bp of 5'-untranslated sequence, 3510 bp of coding sequence, and 687 bp of 3'-untranslated sequence, including a 100-bp poly(A) tail (Fig. 2Go). This sequence has been deposited in the EMBL Nucleotide Sequence Database (accession no. X 96540). A second round of 3'-RACE-PCR using a primer chosen from the pAC1 sequence was performed to produce a clone (pAC5; 2 kb) that overlaps the pAC1 sequence and the 3'-region of the longer TSP2 message. However, as shown in Fig. 1BGo, a set of primers (shown as arrows) that amplified a 300-bp fragment from pAC1 amplified a 1.2-kb fragment from the second clone, pAC5. Both of these fragments could be amplified when cDNA was used as a template (lane RT+), but only the 1.2-kb product could be amplified from genomic DNA (lane Gen), suggesting that the smaller fragment resulted from an alternate splice. This is supported by the finding that probes 1 and 2 (shown in Fig. 1BGo) hybridize to both messages on Northern blots, whereas probe 3 recognizes only the longer message (data not shown). The clone pAC5, therefore, represents the 3'-UTR of the longer transcript.



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  		Figure 3. TSP2 mRNA expression in bovine adrenal tissue and in primary cultures of BAC cells. Total RNAs (20 µg) from bovine adrenal zona glomerulosa (G), zona fasciculata (F), and adrenal medulla (M) tissue slices or control (Ctl) and ACTH-treated (ACTH) BAC cells were run on a 1% agarose-formaldehyde gel and transferred to a nylon membrane as described in Materials and Methods. The membrane was sequentially hybridized with 32P-labeled cDNA probes for bovine TSP2 (upper panels) and for the ribosomal 18S subunit (lower panels), as described in Materials and Methods. Radiolabeled bands were visualized and quantitated using a ß-imager, and their sizes are indicated.

 


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  		Figure 2. Amino acid sequence of bovine TSP2. The amino acid sequence of bovine TSP2 was deduced from the overlapping cDNA sequences. This was then compared with the protein sequences of human (39 ) and mouse (48 49 ) TSP2 using the Clustal W algorithm. Amino acid residues identical in at least two sequences are darkly shaded, and similar amino acid residues are lightly shaded. Dashes denote amino acid gaps. Repeated domains are delimited with brackets, with the beginning of each subrepeat shown by square dots. The remarkable consensus sequences presented in Table 1Go are underlined by various types of bars. The nt sequence of bovine TSP2 was deposited in the EMBL Nucleotide Sequence Database under accession no. X96540.

 
Comparison of CISP sequence with other TSP2 sequences
Figure 2Go compares the derived amino acid sequence of bovine CISP with the published sequences of human and mouse TSP2. All three sequences align almost perfectly, except for a 2-amino acid gap in the bovine sequence corresponding to residues 377–378 in the human sequence. There is striking amino acid conservation between TSP2 from all 3 species, as evidenced by the identical position of the 58 cysteine residues. Bovine TSP2 is 89% identical to human TSP2, 87% identical to mouse TSP2, and 79% identical to chicken TSP2 (data not shown), whereas it is only 61% identical to bovine TSP1 (data not shown). The 5'-noncoding region of bovine TSP2 cDNA is 500 bp long, which is twice as long as that of human (239 bp) or mouse (218 bp) TSP2. In human and mouse, the 3'-UTR is transcribed from a single, approximately 2-kb exon (18). If this is the case for the bovine sequence, then this exon would be alternately spliced to yield a short, 0.6-kb 3'-UTR or a 2-kb sequence similar to that in human or mouse. The bovine 3'-UTR sequence shows homology (59% identity in the first 600 nt) with the 3'-UTR of human TSP2. Our results definitively establish that CISP is the bovine ortholog of TSP2.

Bovine TSP2 messenger RNA (mRNA) is expressed in both adrenocortical tissue and cultured cells
We have previously shown that TSP2 is expressed in BAC cells in culture and can be up-regulated by ACTH treatment (3, 16). To confirm that this expression was not merely an artifact of tissue culture, we examined TSP2 expression in adrenal tissue. As shown in Fig. 3Go, two transcripts at approximately 4.7 and 6.0 kb were detected on Northern blots of total RNA from both adrenal tissue slices and cultured cells. The signal was quantitated via ß-imager and was corrected against the 18S ribosomal RNA as a loading control. Expression was strongest in zona fasciculata tissue (lane F), but was also detectable in zona glomerulosa (lane G) and adrenal medulla (lane M). TSP2 is expressed at similar levels in zona fasciculata tissue and in ACTH-deprived cultured cells (lane Ctrl); less than 2-fold more TSP is expressed in cultured cells (data not shown). The 6-kb transcript accounted for approximately 70% of the TSP2 message in all cell types. Both transcripts were induced by ACTH treatment (lane ACTH; ~9-fold for the 6-kb transcript and 7.7-fold for the 4.7-kb transcript after 12 h of stimulation).

TSP2 is expressed as a 180-kDa protein in several adult bovine tissues
We analyzed the distribution of TSP2 protein in several bovine tissues by Western blotting using polyclonal rabbit anti-bovine TSP2 antibodies. As shown in Fig. 5Go, an immunoreactive band of 180K Mr was clearly detectable in the extracts from adrenal cortex, heart, spleen, brain, kidney, placenta, ovary, and testis. A much weaker 180-kDa band was detected in adrenal medulla, muscle, and liver extracts. In contrast, no signal was visible in intestine or thyroid extracts (Fig. 4Go) or in plasma, serum, or platelet extracts (data not shown). The 180-kDa immunoreactive protein clearly migrated faster than the bTSP2 protein present in the conditioned medium of ACTH-treated BAC cells (apparent Mr of 195K) (3). This protein size difference could result from differential glycosylation or in vivo peptide cleavage.



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  		Figure 5. Variable degrees of N-glycosylation of TSP2 from bovine adrenal cortex or cultured cells. A, Purified bovine TSP2 (2.5 µg), 120 µg cytosolic proteins from bovine adrenal cortex tissue (Ad. cortex), and 2.5 µg purified recombinant mouse TSP2 (rmTSP2) were separated by 6% SDS-PAGE and transferred onto nitrocellulose. TSP2 was then immunodetected by Western blot using either anti-bTSP2 or anti-mTSP2 antibodies. The positions and sizes of molecular mass markers are indicated on the right. B, Purified bovine TSP2 (2.5 µg) and 2.5 µg recombinant mTSP2 (rmTSP2) were incubated overnight in the presence (+) or absence (-) of peptide N-glycosidase F. The untreated and deglycosylated proteins were then analyzed by 6% PAGE-SDS and visualized by Western blot using either polyclonal anti-bTSP2 or polyclonal anti-mTSP2 antibodies. The positions and sizes of molecular mass markers are indicated on the right.

 


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  		Figure 4. TSP2 protein expression in bovine organs. One hundred micrograms of cytosolic proteins from various bovine organ extracts were separated by 8% SDS-PAGE and transferred onto nitrocellulose as described in Materials and Methods. Two milliliters of conditioned medium from BAC cells treated for 24 h with 10-8 M ACTH were trichloroacetic acid precipitated and run in a parallel lane as a control. TSP2 was then detected by Western blot using rabbit polyclonal antibodies against bovine TSP2. The position and size (in kilodaltons) of molecular mass markers are indicated on the left. The sizes of immunoreactive bands are indicated on the right. Ad.C., Adrenal cortex; Ad.M., adrenal medulla; He, heart; Sp, spleen; In, intestine; Br, brain; Ki, kidney; Pl, placenta; Ov, ovary; Te, testis; Th, thymus; Mu, skeletal muscle; Li, liver; BAC/ACTH, conditioned medium from ACTH-treated BAC cells.

 
Chen et al. previously reported that the monomer of recombinant mouse TSP2 expressed in insect cells also has an apparent Mr of 180 kDa (13). This prompted us to compare the SDS-PAGE migration of bovine TSP2 from adrenal cortex tissue and BAC cell-conditioned medium with that of recombinant mouse TSP2 produced by baculovirus-infected insect cells. Recombinant mouse TSP2 and immunodetectable TSP2 from adrenocortical tissue comigrated as 180-kDa bands, whereas purified TSP2 from BAC cell-conditioned medium migrated as a 195-kDa band (Fig. 5AGo). We digested these proteins with peptide N-glycosidase F to remove N-linked oligosaccharide chains from the core proteins. Our analysis did not include TSP2 from adrenal cortex tissue because we were not successful in fully deglycosylating it due to degrading proteolysis that occurred during the long incubation period, even in the presence of a cocktail of protease inhibitors. As observed in Fig. 5BGo, deglycosylation of the 180-kDa recombinant mouse TSP2 reduced its apparent Mr to 175K, whereas deglycosylation of the 195-kDa bovine TSP2 reduced its apparent Mr to 180K. Bovine TSP2 made by adrenocortical cells in primary culture thus appears to be more extensively N-glycosylated than recombinant mouse TSP2 made by insect cells.

TSP2 is present in the external zones of the bovine adrenal cortex
We immunolocalized TSP2 in sagittal sections of bovine adrenal glands using a polyclonal rabbit antibovine TSP2 antibody developed in our laboratory. The results presented in Fig. 6Go show abundant immunoreactive signal in the adrenocytes from the glomerulosa and fasciculata zones. A decreasing gradient of staining was observed extending from the inner fasciculata zone toward the reticularis zone. Some cells in the external part of the adrenal medulla were stained, whereas the cells of the inner medulla and the adrenal capsule were negative.



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  		Figure 6. Immunolocalization of TSP2 in the bovine adrenal gland. A, Sections of bovine adrenal glands were immunostained with either anti-bTSP2 IgGs or nonimmune IgGs as described in Materials and Methods. Strong TSP2 immunoreactivity was observed in the glomerulosa and fasciculata zones of the cortex. A weaker signal was observed in the reticularis zone of the cortex and in the chromaffin cells of the medulla. Magnification, x20. B, Larger magnification (x400) of the individual zones.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ACTH is the major regulator of adrenal cortex development and function. Besides its well characterized acute and chronic effects on corticosteroidogenesis, it also drives the histological organization of this tissue (19, 20). After ACTH deprivation caused by hypophysectomy, the adrenal cortex dramatically regresses to a thickened glomerulosa zone (21). ACTH reperfusion allows the reconstruction of a fully vascularized and differentiated tissue, with a zonal organization identical to that found before hypophysectomy. This indicates that ACTH directs a number of distinct biological functions, ranging from cell migration to cell differentiation, cell proliferation, and cell death. We hypothesized that a panel of ACTH-regulated extracellular proteins exists that mediates these various effects. Comparative analysis of the protein composition of conditioned medium from control and ACTH-treated fasciculata-reticularis adrenocytes led to the identification of a large trimeric glycoprotein (Mr = 3 x 195K) whose concentration in culture medium is increased 5- to 14-fold upon ACTH treatment (3). In the present study, we report the isolation of overlapping cDNA clones that encode the full-length CISP. Alignment of the deduced protein sequence with published TSP sequences revealed 89% identity to human TSP2, but only 61% identity to bovine TSP1, thus allowing us to definitively establish that CISP is the bovine ortholog of TSP2.

The bovine TSP2 sequence aligns perfectly with the human and mouse TSP2 sequences, except for a gap of two amino acids. This deletion is located in the linker region between the procollagen module and the first type I repeat. It is thus not expected to have major consequences on the functions of the protein. This deletion seems to be a feature of bovine TSP2 rather than a polymorphism, as these residues are also missing in a partial cDNA for bTSP2 independently isolated by another team (EMBL Nucleotide Sequence Database, accession no. X87620). In contrast, the second gap of one amino acid (Asp743 in the human sequence) that was observed in one of the RACE-PCR cDNA clones that we sequenced probably reflects a polymorphism, as the missing Asp codon was present in a second RACE cDNA clone and in the sequence deposited by the other team (no. X87620). This polymorphism changes the length of an acidic cluster of aspartate residues located in the second type III repeat from five to four residues. At present, it is not known whether this polymorphism has any consequence on the protein’s functions. However, an intriguingly similar 3-bp deletion resulting in the loss of one of the five successive aspartate residues constituting a calcium-binding site in one of the type III repeats of COMP/TSP5 has been found in five unrelated patients with pseudoachondroplasia (22).

As shown in Table 1Go, most of the functional motifs present in other TSP1 and TSP2 molecules are conserved in bovine TSP2. These include the heparin-binding WSPW motifs (23, 24), the CD36-binding CSVTCG repeats (25, 26), and the fibronectin-binding GGWSHW sequence (27).


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  		Table 1. Active peptide sequences in TSPs

 
Murphy-Ullrich and colleagues have reported that TSP1 is able to activate latent transforming growth factor-ß (TGFß) (28). They identified the tripeptide RFK from the type I repeats of TSP1 as a sequence sufficient to trigger this activation (29). They also stated that recombinant mouse TSP2 was unable to activate latent TGFß due to the substitution in human TSP2 of RIR for the RFK motif. In contrast, we previously reported that bovine TSP2 is able to activate partially purified large latent TGFß complex (30), but is unable to form a molecular complex with or activate recombinant small latent TGFß (31). Although bovine TSP2 possesses an RIR sequence identical to that of TSP2 from other species, it should be noted that bovine, human, and murine TSP2 all have a common KKFK sequence located in the procollagen domain (344–347) that could substitute for the activating KRFK sequence of TSP1. It will be of interest to determine whether this peptide is potent in activating latent TGFß.

Another important biological function of TSPs is their potent capacity to inhibit angiogenesis. Both TSP1 and TSP2 inhibit in vitro migration of capillary endothelial cells toward a variety of inducers (ranging from PGs to vascular endothelial growth factor, TGFß, and fibroblast growth factor-2) as well as block fibroblast growth factor-2-stimulated capillary outgrowth in the rat cornea (32). TSP1 inhibits capillary endothelial cell proliferation, migration, and tube formation and thus appears to block the entire program of dedifferentiation and redifferentiation essential to neoangiogenesis (33). The antiangiogenic activity of TSP1 is mimicked by the peptide sequences NGVQYRN from the procollagen homology domain and two SPWXXCSVTCG sequences from the type I repeats (34). Binding of these two sequences to CD36 appears to mediate the angiostatic activity of TSP1 (35). In bovine and human TSP2, the first motif (DGRFFA) is quite different from the corresponding procollagen TSP1 motif, whereas the motifs from the type I repeats are mostly conserved: SPWSSCSVTCG and SPWSACTVTCA. This suggests that these type I repeat motifs are more important than the NGVQYRN motif in mediating the antiangiogenic activity of TSPs.

Two TSP2 mRNAs of 6.0 and 4.7 kb are expressed in the bovine adrenal cortex and adrenal medulla in an approximately 2:1 ratio. Both transcripts encode the full-length protein, and the difference in size appears to be the result of an alternative splice in the 3'-UTR. Both transcripts are regulated by ACTH in a similar manner (8- to 9-fold induction). In human tissues, a major TSP2 transcript of 7.5 kb has been reported, but a minor 6.0-kb transcript is also visible on Northern blots of smooth muscle cells and the osteosarcoma cell line MG63 (36). In contrast, a single transcript of 6.0 kb has been described in mouse tissues (37). It is possible that the two bovine TSP2 transcripts differ in stability, as two poly(T) stretches have been identified in the 3'-UTR of TSP2 that could protect the mRNA from 3'-exonucleolytic attack by forming a loop with the poly(A) tail (38).

Mature TSP2 from adrenal cortex tissue (~180 kDa) and adrenocortical cell cultures (~195 kDa) differ in size. Interestingly, the apparent Mr of recombinant mouse TSP2 produced in baculovirus-infected Sf9 insect cells was similar to that of tissue-derived bovine TSP2. Deglycosylation of recombinant mouse TSP2 and purified bovine TSP2 from culture medium using peptide N-glycosidase revealed that these two proteins are N-glycosylated to different extents (removal of 3–5 kDa from mouse TSP2 vs. removal of 15 kDa from bovine TSP2). However, the sizes of the deglycosylated proteins still differed by 3–5 kDa. As human, mouse, chicken, and bovine TSP2s differ in length by no more than six amino acids (39, 40), the slight molecular size difference between the two deglycosylated proteins could be due to O-glycosylation occurring only on the bovine protein. The facts that bovine TSP1 has been shown to be O-glycosylated (41) and that this posttranslational modification is carried out poorly in insect cells (42) would support this hypothesis.

The distribution of TSP2 among adult bovine tissues appears to be widespread, although not ubiquitous. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) express TSP2, with the exception of the thyroid gland. TSP2 is undetectable in skeletal muscle, intestine, or serum, but it is weakly expressed in the liver and adrenal medulla. After the adrenal cortex, the heart, spleen, brain, and kidney contain the highest levels of TSP2 protein. Thus, TSP2 expression is more widespread among adult tissues than is TSP1 expression (43). This was also observed in the 18-day-old mouse embryo using in situ hybridization (44). In this latter study, most tissues, except skin, were found to express TSP2. It will be interesting to determine whether TSP2 synthesis is stimulated by cAMP or cAMP-inducing hormones (such as FSH, LH, or TSH) in these tissues to the same extent as it is by ACTH in the adrenal cortex. The results of TSP2 gene invalidation in mice are indicative of multiple sites of action. TSP2-null mice present disordered collagen fibers in skin and tendons, increased bone thickness, bleeding diathesis, and an increase in blood vessel density in several tissues (45). This last phenotype is in agreement with a major role of TSP2 as an angiostatic factor.

The biological function of TSP2 in the adrenal cortex for the most part remains elusive. We have observed some biological effects in vitro. Coating of tissue culture dishes with bTSP2 stimulates the adhesion, but prevents the spreading of adrenocortical fasciculata cells (46). TSP2 was also reported to sustain the rounding-up of adrenocortical cells induced by ACTH treatment, as the addition of anti-TSP2 antibodies dramatically reduced the duration of this morphological change (16). These observations are difficult to correlate with any in vivo effect of ACTH. No major alteration in corticosteroid hormone secretion or adrenal tissue histology was observed in TSP2-null mice (Kyriakides, T., and P. Bornstein, personal communication), indicating that TSP2 is not essential for or may be replaced by a functionally redundant protein in adrenal cortex development.

To gain insight into the in vivo function of TSP2, we examined its distribution in the intact gland. The expression of both the 4.7- and 6.0-kb TSP2 mRNAs was higher in the adrenal cortex than in the adrenal medulla. Compared with the mRNA levels in primary cultures of bovine adrenocortical fasciculata cells, the abundance of TSP2 mRNA in the adrenal cortex was similar to that in control cultures, but was much lower than that in ACTH-treated cultures. This suggests either that only a small subpopulation of adrenocortical cells produce TSP2 in response to ACTH in vivo or that the physiological levels of ACTH (~10-11 M) suboptimally stimulate TSP2 synthesis. Both factors probably contribute to the relatively low abundance of TSP2 mRNA in vivo. Immunohistochemical analysis showed that TSP2 is strongly expressed in the glomerulosa and fasciculata zones, but is very weakly expressed in the reticularis zone. This pattern of expression is superimposable with the reported zonal distribution of ACTH receptors in the adrenal cortex of primates (47), suggesting that ACTH is the main regulator of TSP2 expression in vivo. It is thus reasonable to consider TSP2 as a candidate relay protein of ACTH for a subset of its biological actions in the adrenal cortex.


   Acknowledgments
 
We are indebted to Claude Blanc-Brude, Naïg Le Guern, and Isabelle Gaillard for their help in the preparation of primary cultures of bovine adrenocortical cells. We thank Dr. Sabine Bailly for critical reading of the manuscript. We acknowledge the excellent secretarial help of Sonia Lidy and Patricia Gabriel.


   Footnotes
 
1 This work was supported by INSERM, the Commissariat à l’Energie Atomique (CEA/DSV/DBMS/BRCE), the Ligue Nationale contre le Cancer, and postdoctoral fellowships from INSERM (to M.D. and A.M.C.). Part of this work was supported by an INSERM/FRSQ cooperative research program. Back

2 M.D. and A.M.C. contributed equally to this paper. Back

3 Present address: Douglas Hospital Research Center, McGill University, 6875 boulevard La Salle, Verdun, Québec, Canada. Back

4 Present address: Centre de Transfusion Sanguine de la Loire, 25 boulevard Pasteur, 42000 Saint-Etienne, France. Back

5 Present address: INSERM U-271, 151 cours Albert Thomas, 69000 Lyon, France. Back

6 Present address: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215. Back

Received April 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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