ARTICLE |
Correspondence to: E. Helene Sage, Dept. of Biological Structure, Box 357420, U. of Washington, Seattle, WA 98195-7420.
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Summary |
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A number of cDNAs (SC1, QR1, and hevin) have been shown to be similar to SPARC (secreted protein acidic and rich in cysteine), a matricellular protein that regulates cell adhesion, cell cycle, and matrix assembly and remodeling. These proteins are 61-65% identical in the final 200 residues of their C-termini; their N-terminal sequences are related but more divergent. All have an overall acidic pI, with a follistatin-like region that is rich in cysteine, and a Ca+2 binding consensus sequence at the C-terminus. Using degenerate primers representing the most highly conserved region in SPARC, SC1, and QR1, we identified a 300-BP SC1 clone in a primary polymerase chain reaction (PCR) screen of a mouse brain cDNA library. This cDNA was used to obtain a full-length clone, which hybridized to a 2.8-KB RNA abundant in brain. Mouse SC1 displays a similarity of 70% to mouse SPARC at the amino acid level. Northern blot and RNAse protection assays revealed a 2.8-KB mRNA expressed at moderate levels (relative to brain) in mouse heart, adrenal gland, epididymis, and lung, and at low levels in kidney, eye, liver, spleen, submandibular gland, and testis. In contrast to SPARC, in situ hybridization showed expression of SC1 mRNA in the tunica media and/or adventitia of medium and large vessels; transcripts were not detected in capillaries, venules, or large lymphatics. The distribution of transcripts for SC1 was also different from that of SPARC in several organs, including adrenal gland, lung, heart, liver, and spleen. Moreover, SC1 mRNA was not evident in endothelium cultured from rat heart, bovine fetal and adult aorta, mouse aorta, human omentum, and bovine retina. Cultured smooth muscle cells and fibroblasts also failed to express SC1 mRNA. The absence of SC1 transcript in cultured cells indicates that the SC1 gene is potentially sensitive to regulatory factors in serum or to a three-dimensional architecture conferred by the extracellular matrix that is lacking in vitro. In conclusion, the expression of SPARC and SC1 appears to be coincident in specific tissues (e.g., adrenal gland and brain), but these proteins exhibit distinct expression patterns in most organs of the mouse. Because SC1 and SPARC are structurally similar and exhibit counteradhesive effects on cultured cells, their overlapping and/or adjacent expression in most tissues predicts that one protein might compensate functionally, at least in part, for the other. The sequence reported here has been deposited in the GenBank data base (accession no. U77330). (J Histochem Cytochem 45:823-835, 1997)
Key Words: SPARC, SC1, hevin, development, mouse
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Introduction |
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SPARC (OSTEONECTIN, BM-40), an extracellular matrix-associated glycoprotein, is expressed in a variety of tissues during embryogenesis and wound repair (reviewed in
SPARC is a member of a family of proteins that exhibit a similar basic structure. These proteins have clusters of acidic residues imparting an overall acidic pI, a follistatin-like region that is cysteine-rich, and a high-affinity Ca2+ binding region termed an EF hand (
In this study we used polymerase chain reaction (PCR) methods, in addition to library screening, to search for other SPARC family members. We describe the cloning of SC1 from a mouse whole-brain cDNA library. We subsequently used ribonuclease protection and in situ hybridization assays to examine the distribution of mouse SC1 mRNA in various organs and during development. Our results indicate that the primary site of expression of SC1 mRNA is the brain, although lower levels of transcript were noted in several other organs/tissues. In the vasculature, SC1 was associated with the adventitia and/or media of medium and large vessels in all organs examined. Because SPARC and hevin perform overlapping functions in vitro, it is possible that these proteins compensate for each other during development or response to injury.
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Materials and Methods |
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Isolation of SC1 cDNA from Mouse Whole-brain Library
Degenerate primers designed according to the C-terminal regions of SPARC, QR1, and rat SC1 (Figure 1) were used in a primary screen of a mouse whole-brain cDNA library (gift from Jim Boulter, Salk Institute) by the MOPAC procedure as described by 32P]-ATP (New England Nuclear; Boston, MA), 5' exchange buffer, and polynucleotide kinase (Gibco BRL; Grand Island, NY). Bands that hybridized with the probes were isolated on 4% NuSieve gel and were excised from the gel. The agarose was subsequently removed with Agarase (Boehringer Mannheim Biochemicals; Indianapolis, IN). These cDNA clones were subcloned into a blunted Eco RV site in a Bluescript II SK (+/-) vector (Stratagene) and were transformed into competent XLI-Blue bacteria. Positive clones were selected and were cultured in Luria broth. Pure DNA was isolated by mini prep (Qiagen; Chatsworth, CA), and clones were sequenced in conjunction with T7 and T3 primers (Stratagene) according to the procedure of
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Positive clones from the primary screen were used as probes to screen the cDNA library for full-length clones. The library was plated at 5000 pfu/ml. Probes were random-prime labeled (Amersham; Arlington Heights, IL) with [32P]-dCTP (New England Nuclear). Duplicate filter lifts (Amersham) from 20 plates, each containing 50,000 plaques, were hybridized overnight at 42C in 3 x SSC [1 x SSC is 0.15 M NaCl, 0.015M Na-citrate (pH 7.5)], 0.5% SDS (sodium dodecyl sulfate), 25 mM Tris-HCl, 0.2% Ficoll, 0.2% polyvinylpyrrolidone (MW 40,000), 0.2% BSA (bovine serum albumin), and 100 µg/ml salmon sperm DNA. The membranes were next washed with 1 x SSC, 0.1% SDS at room temperature (four times for 20 min) and 0.1 x SSC, 0.1% SDS at 50C (twice for 20 min). Positive plaques were selected and re-screened until all plaques on a plate were positive. These plaques were selected, and the corresponding pBluescript I (SK+) phagemids were rescued by excision in vivo with VCS M13 helper phage. Inserts were released from plasmid DNA by digestion with restriction enzymes EcoRI and XhoI, separated on a 1% agarose gel, stained with ethidium bromide, and transferred to Duralose membranes. Membranes were probed with the same probe used to screen the library. Positive clones were sequenced with an ABI 373A automated sequencer (Applied Biosystems; Foster City, CA). The GCG and BLASTX programs were used to analyze the sequences.
5' RACE (rapid amplification of cDNA ends) (32P]-dCTP (New England Nuclear) and was hybridized to the membrane. Positive reaction products were cloned into the TA vector (Invitrogen). Clones were sequenced on an automated sequencing machine.
RNA Extraction and Northern Analysis
Total RNA was isolated from mouse tissues (C57BL/6J) and cultured cells with Tri-Reagent (Molecular Research Center; Cincinnati, OH). Samples were resuspended in water treated with diethyl pyrocarbonate (DEPC) (Sigma; St Louis, MO), and the concentration of RNA was calculated by UV absorbance.
Mouse SC1 expression was analyzed by Northern blotting. An 800-BP fragment from the N-terminus of the mouse SC1 cDNA clone was random prime-labeled (Amersham) with [32P]-dCTP (New England Nuclear) and was used as a probe. The filters were stripped with two washes of boiling 0.1% SDS (10 min each) and were subsequently hybridized with a 570-BP fragment from the N-terminus of mouse SPARC cDNA, followed by hybridization with a 28S rRNA probe for normalization of signals. Autoradiographs were analyzed by scanning densitometry with a DU-70 Spectrophotometer (Beckman Instruments; Palo Alto, CA).
Ribonuclease Protection Assays
An 800-BP EcoRI fragment of mouse SC1 was subcloned into Bluescript SK+ (Stratagene) and was linearized with Ear I for use in riboprobe labeling. A 570-BP EcoRI fragment of mouse SPARC was also subcloned into pBluescript SK+ and was linearized with BamHI for use in riboprobe labeling. SC1, SPARC, and 28S (Invitrogen) riboprobes were transcribed with an RNA transcription kit (Promega; Madison, WI) in the presence of [32P]-UTP (New England Nuclear) with T3, T7, and SP6 RNA polymerases, respectively. The mouse SC1 riboprobe is expected to protect a 360-BP fragment, SPARC riboprobe a 570-BP fragment, and 28S riboprobe (182 BP) a 115-BP fragment. The probes were purified on a 6% acrylamide gel.
RNAse protection assays were performed as previously described (
In Situ Hybridization
The mouse SC1 and SPARC riboprobes were transcribed with an RNA transcription kit (Promega) in the presence of [35S]-dUTP (Amersham). The specific activity of the probes was approximately 3-4 x 107 cpm/µg. The riboprobes were transcribed from a 800-BP EcoRI mouse SC1 cDNA fragment, cloned in an anti-sense orientation relative to the T3 promoter in a pBluescript SK+ vector, and from a 1147-BP BamHI/HindIII mouse SPARC cDNA fragment, cloned in anti-sense orientation relative to the SP6 promoter in a pGEM-1 vector. The respective riboprobes, transcribed in a sense orientation, were used for control experiments.
The in situ hybridization protocol was a modification of a method described by
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Results |
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Identification and Cloning of Mouse SC1
Degenerate oligonucleotide primers designed against the conserved C-terminal regions of mouse SPARC, quail QR1, and rat SC1 were used in a PCR screen of a mouse whole-brain cDNA library. Figure 1 shows the location of these primers in the amino acid sequence of these proteins. Isolated clones were sequenced and were compared to SPARC, QR1, and SC1. Clones that showed some similarity (80% or greater at the nucleotide level) to any of the three proteins were used to re-screen the library to retrieve the full-length cDNA. One of the clones isolated in this manner was identified as mouse SC1. The clone was lacking about 800 BP of its most 5' region, including the methionine start site; 5' RACE was utilized to obtain the rest of the clone. These fragments were subsequently ligated at an overlapping Bam HI site.
An 800-BP fragment from the N-terminal region of the clone was used to probe a Northern blot of 2 µg of whole brain poly A+ mRNA. A single mRNA of 2.8 KB was recognized by the probe (data not shown). The full-length mouse SC1 is 2797 BP, with a coding sequence of 1950 BP (Figure 2). The mouse SC1 cDNA contains a single open reading frame that encodes a protein sequence of 650 amino acids. The N-terminal end contains a sequence of 18 amino acids that is highly hydrophobic and could represent a signal peptide. Figure 3 shows a comparison of the mouse SC1 amino acid sequence with those of rat SC1 and (human) hevin. Mouse SC1 has a similarity of 93% at the amino acid level to rat SC1 and of 77% to hevin, as well as a similarity of 70% to mouse SPARC.
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Expression of Mouse SC1 and SPARC in Different Tissues
Ribonuclease protection assays were performed with riboprobes for the N-terminal regions of SPARC and mouse SC1 on total RNA isolated from brain, heart, kidney, adrenal gland, lung, liver, eye, submandibular gland, epididymis, and testis. The expression pattern for these two mRNAs was similar (Figure 4A and Figure 4B). Figure 5 is a graphic representation of the data shown in Figure 4. Compared to SC1 mRNA seen in brain, expression in other tissues was 60-90% lower in abundance. To ascertain whether SPARC and SC1 mRNAs were expressed by the same cell types, we performed Northern blots on a variety of cultured endothelial cell types (rat heart endothelium, bovine fetal and adult aorta, mouse aorta, human omentum, human aortic, and bovine retinal endothelial cells), as well as on smooth muscle cells and fibroblasts from mouse, bovine, and human sources. Although SPARC transcripts were abundant in these cells, SC1 was not expressed at detectable levels by endothelial cells, smooth muscle cells, or fibroblasts (data not shown).
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In Situ Hybridization Assays in Mouse Tissues
In situ hybridization was performed to determine the distribution of SC1 mRNA. Figure 6 shows in situ hybridization on rat kidney and adrenal gland and on mouse kidney. Figure 6A-C represent tissues probed with anti-sense SPARC mRNA and Figure 6D-F represent serial tissue sections probed with anti-sense SC1 mRNA. SPARC mRNA was found in the glomerular capillaries (Figure 6A and Figure 6C) of the kidney, as well as in medium and large vessels. Expression of SPARC mRNA in the adrenal gland (Figure 6B) was predominant in the zona fasciculata of the adrenal cortex, with lesser amounts in the zona glomerulosa, zona reticularis, and medulla. Expression of SC1 mRNA in the kidney (Figure 6D and Figure 6F) was also localized to the medium and large vessels of the kidney, whereas in the adrenal gland (Figure 6E), low levels of transcripts were detected throughout all zones of the cortex and in the medulla.
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Figure 7 shows SC1 mRNA in heart, lung, liver, spleen, and epididymis. In the heart (Figure 7A), transcripts are noted in medium and larger-sized vessels coursing through the cardiac muscle and in a localized area of the apex consistent with terminal fibers of the conducting tissue of the heart. SC1 mRNA in the lung (Figure 7B) was detected in medium and large vessels and in the bronchi and bronchioles. The liver (Figure 7C) showed expression of SC1 mRNA almost exclusively in medium and larger vessels. Transcripts were not detected in the sinuses of the liver (Figure 7C) or the spleen (Figure 7D). In the spleen, SC1 mRNA was scattered throughout the red pulp. Transcripts were absent in the white pulp except for the expression of SC1 mRNA in medium-sized blood vessels (Figure 7D). In the epididymis (Figure 7E), transcripts were present in the vessels surrounding the tubules and in cells of the interstitia. Figure 7F represents the liver hybridized with the sense SC1 riboprobe.
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Highly vascular organs were studied to examine further the expression of SC1 in vessels of different sizes. In all tissues examined, expression of SC1 mRNA in spleen and liver was observed in the media and adventitia of medium and large vessels but not in the endothelial cell layer (Figure 8). Figure 8A shows that, in the spleen, the endothelial cells were devoid of SC1 transcript, but the media and extracellular matrix surrounding arterioles contained relatively high levels of mRNA. Moreover, no transcripts were detected in the sinus adjacent to the arteriole. Figure 8B shows the concentration of signal in the media and adventitia of the vessel but not in the endothelial cell (intimal) layer or the adjacent sinus of the liver. In contrast, expression of SC1 mRNA was generally negative in small arterioles and venules of the liver (Figure 8C). SC1 transcripts were present at low levels throughout the capsule, medium-sized vessels, and parenchyma of lymph nodes (data not shown). Small vessels of the lymph node, such as the high endothelial venules, were not distinguishable, and localized transcripts were, therefore, difficult to detect.
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Mouse embryos from various stages of development were examined to determine whether the distribution of SC1 was regulated in a differential manner during embryogenesis (Figure 9). At Day 12 (Figure 9A), SC1 mRNA was diffuse throughout the central and peripheral nervous system and in developing bone. Similar levels of expression were noted in the Day 13 embryo (Figure 9B-C). Figure 9D represents the brain of a Day 15 embryo. As noted during other time points of development, medium and large-sized vessels as well as the meninges of the brain expressed SC1 mRNA. Transcripts for SC1 were also noted throughout the brain parenchyma. At Day 16, SC1 transcripts were also prominent in the lung, interstitial tissue, and again in medium and large vessels (Figure 9E). Expression in many organs, such as the heart and liver, was limited to the capsules surrounding the organs and larger blood vessels (Figure 9E). In the gut, abundant transcripts were noted in the mesentery, the vasculature, and in many of the epithelial cells (Figure 9E). As noted earlier in development, there were marked levels of expression in the spinal cord and in the developing bone of the vertebrae (Figure 9F). In the Day 18 embryo (Figure 9G-I), SC1 mRNA was abundant in the area of the sensory nerve supply and surrounding connective tissue of the vibrissae (Figure 9G), in the spinal cord, and in the gut (Figure 9H). Bone islands, such as those of the foot, continued to express abundant transcripts for SC1 (Figure 9I).
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Discussion |
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Members of the SPARC family include albondin, follistatin-like protein (TSC-36), testican, QR1, hevin, and SC1 (
SC1 is thought to play an important role in neural development.
Hevin (human SC1) is a protein associated with the basal, lateral, and apical surfaces of human high endothelial venule (HEV) cells and is not expressed in the underlying basement membrane (
Both SC1 and SPARC are expressed in many organs in the mouse. Ribonuclease protection assays showed similarities in tissue expression but notable differences in relative abundance between these two mRNAs. Moreover, in situ hybridization demonstrated that transcripts for these two proteins were not expressed in the same regions of a particular organ or by the same cell type. For example, SC1 was expressed almost exclusively in the medium and large vessels of the kidney, whereas SPARC transcript was seen in the glomeruli as well as the vasculature of the kidney. During development, patterns of expression of SC1 were similar to those found in the adult mouse, with prominent levels in the brain and spinal cord, and in the media and adventitia of vessels. After the development of the gut, the mesenteric membrane of the small intestine showed abundant transcripts for SC1 mRNA. This supporting structure is composed of fibrous tissue containing a network of fibroblasts and mesothelial cells that synthesize a number of ECM proteins, including collagens and elastin. In spite of the high levels of expression in cells that provide structural support in vivo, cultured smooth muscle cells and fibroblasts isolated from mouse aorta had no detectable SC1 mRNA. Conditions conducive to transcription of SC1 mRNA, which might be lacking in vitro, include the presence of specific extracellular matrix components and/or growth factors and a three-dimensional network.
Although SC1 and SPARC share a similar pattern of tissue distribution, they are not found in the same structures within organs. However, the cell adhesion studies done by
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Acknowledgments |
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Supported by the National Institutes of Health (GM40711).
We thank Dr L. Isom for guidance in the initial experimental design of this project, and S. Soderling and Dr K. Motamed for helpful discussions. We also thank Dr J. Boulter of the Salk Institute for his generous gift of cDNA libraries.
Received for publication November 7, 1996; accepted January 16, 1997.
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