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Ruijin Hospital (S.-M.H., H.-M.Y., R.-Y.L., Q.-Y.M., T.-J.Y., J.-L.C., H.-D.S.), State Key Laboratory of Medical Genomics, Endocrine and Metabolic Division, E-Institutes of Shanghai Universities, Shanghai Institute of Endocrinology; and Departments of Medical Genetics (S.-M.H., Z.-Y.L.) and Biochemistry and Molecular Biology (F.L.), Shanghai Second Medical University, Shanghai, China, 200025
Address all correspondence and requests for reprints to: Dr. Huai-Dong Song, Ruijin Hospital, State Key Laboratory of Medical Genomics, Shanghai Second Medical University, Shanghai, China, 200025. E-mail: huaidong_s1966{at}163.com.
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Abstract |
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Objective: We hypothesize that the mimecan gene expressed in the human pituitary and regulated by pituitary transcription factor-1 (Pit-1) might act as a marker for diagnosing pituitary tumors.
Design: The clinical aspect of our work was a cross-sectional study.
Setting and Patients: In total, 20 pituitary tumor samples were collected from January 1, 2002, to December 30, 2002, in Ruijin Hospital, Shanghai, China.
Intervention: The number of pituitary tumors was limited. Collection of more pituitary tumor samples for additional observation will be necessary.
Main Outcome Measures: The main outcomes were measured by Northern blot, in situ hybridization, immunohistochemical analysis, and so on.
Results: The mimecan gene was expressed at a moderate level in the mouse pituitary gland by Northern blot analysis. Expression of mimecan mRNA and protein is also observed in the human anterior pituitary gland. Luciferase reporter analysis and electrophoretic mobility shift assays show that Pit-1 activates the human mimecan promoter through Pit-1 response element sites. In addition, our data also show that almost all the ACTH- or GH-positive pituitary tumors likely express mimecan protein, and only a portion of prolactin-, TSH-, FSH-, and LH-positive pituitary tumors express mimecan protein.
Conclusions: This work provides insight into the regulating mechanism of mimecan in pituitary and suggests that mimecan may be an unidentified pituitary secretory protein, and certain pituitary cells secreting ACTH or GH also secrete mimecan.
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Introduction |
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The genomic structure of mimecan is highly conserved between species; a single copy of the gene gives rise to multiple mRNA transcripts, which result from differential splicing and alternative polyadenylation (4). However, all mimecan mRNAs produce an identical protein that is conserved between mice, bovine, and human, suggesting its functional importance (3, 5, 6). Mimecan is present in many tissues as a nonsulfated glycoprotein, and multiple mimecan proteins of different sizes have been identified from various tissues. Specifically, a 12-kDa glycoprotein corresponding to the 105 most carboxyl-terminal amino acids of mimecan has been isolated from bovine bone (1, 3, 5), and a 25-kDa keratan sulfate glycoprotein corresponding to the 223 most carboxyl-terminal amino acids of mimecan has been isolated from bovine cornea (3, 7, 8), whereas the full-length 34 kDa protein has been named mimecan (3). It is reasonable to speculate that the multiple-size protein products of this gene appear to result from in situ proteolytic processing.
The physiological functions of mimecan have not been fully understood. Initial studies have shown that mimecan can induce ectopic bone formation; sc implantation of mimecan plus TGF-ß type 1 or 2 into rats induces bone formation at the implantation site (9). However, additional study showed that copurifying bone morphogenetic proteins (BMPs) were the source of its growth-stimulatory activity in this preparation (3). In atherosclerotic lesions, mimecan mRNA was down-regulated in the media and up-regulated in the activated endothelium and thick neointima, whereas the protein accumulated in the front edge of migrating smooth muscle cells (10). More recently, both pro- and mature mimecan have been shown to regulate type I collagen fibrillogenesis, and conversion of mimecan from the precursor to mature form, through cleavage by BMP-1, potentiates the ability of mimecan to modulate the formation of collagen fibrils (11).
To date, there also have been several studies on the transcriptional regulation of the gene encoding mimecan. Growth factors and cytokines can modulate mimecan mRNA expression in corneal keratocytes and vascular smooth muscle cells (12, 13), indicating that mimecan may play a role in cellular growth control. The tumor suppressor protein p53 can activate transcription of the bovine and human mimecan genes (14, 15, 16). Furthermore, human mimecan mRNA is absent or at low levels in a majority of cancer cell lines and tumors, where p53 frequently is inactivated/mutated (14, 15, 16). In addition, the human mimecan promoter contains several UV-responsive regulatory elements that include the intronic p53 DNA binding site and the E-box in the proximal promoter. The E-box plays an important role in transcription and the UV response of the human mimecan promoter. UV irradiation modulates expression of mimecan mRNA in bovine corneal keratocytes and noncorneal cells (17). Recently, it has been shown that mimecan expression in the cornea is suppressed by interferon-
. Interferon regulatory factor-1 (IRF-1) binds to interferon-stimulated response element (RE) sites of the human mimecan gene and negatively regulates mimecan expression at the level of transcription. Consistent with these observations, an inverse correlation between the expression of mimecan and IRF-1 in bovine corneal keratocytes and noncorneal cells has been demonstrated. However, IRF-2 positively regulates mimecan transcription in corneal keratocytes (18).
During our establishment of the gene expression profile in the human pituitary gland, we have identified the full-length cDNA of the mimecan gene (GenBank no. AF100758) (19). In this study, we describe the expression of mimecan in the mouse and human pituitary glands. We also present evidence that pituitary transcription factor-1 (Pit-1), a transcription factor specifically expressed in the pituitary, is a transcriptional activator of the human mimecan promoter. Finally, we found that mimecan protein is expressed in some types of pituitary tumors. Together, these findings provide insights into the regulation and possible function of mimecan in the pituitary.
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Materials and Methods |
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The mouse mimecan gene fragment (530 bp) was amplified by PCR from mouse cDNA and then cloned into pGEM-T Easy vector (Promega, Madison, WI) to generate pGEMT-M-mimecan plasmid used as a probe in Northern blot analysis. Similarly, the human mimecan gene (372 bp) was amplified from human cDNA and cloned into pGEM-T to generate pGEMT-H-mimecan plasmid used in in situ hybridization analysis. A construction, containing full-length Pit-1 cDNA, pGEMT-Pit1 was conserved in our lab. The construct used for in vitro translation of Pit-1, pcDNA3-Pit1, was generated by subcloning the 1.1-kb NotI/SpeI fragment from pGEMT-Pit1 between the NotI and XbaI sites of the pcDNA3 vector.
The reporter plasmid, pGL3/h-mimecan (2.4 kb, pGL3-2.4), containing 2423 bp (–1350 to +1072 bp) of the 5'-flanking region of the human mimecan gene, a gift from Dr. Elena S. Tasheva (Kansas State University, Manhattan, KS), was used as the template for PCR amplifications to generate a series of deletion constructs of the human mimecan promoter. Two different lengths of the 5'-flanking region [1.8 kb (–789/+1072) and 1.5 kb (–468/+1072)] of the human mimecan gene were amplified using the following primers: sense, Hm-789, 5'-GTGCGGTACCGTAAAAGCACCAAGGAGGAAT-3', and Hm-468, 5'-GTGCGGTACCGGAGAAAAAATCCAAATGAC-3'; antisense, Hm-1072, 5'-TTTCTCGAGGGTGTGCGCAGTAAGG-3'. The two sense primers contained a KpnI site, and the antisense primer contained an XhoI site (underlined) at their 5' end to facilitate positional cloning. The resultant DNA fragments were ligated into the KpnI-XhoI site of the pGL3-basic firefly luciferase expression vector (Promega) to generate pGL3-1.8 and pGL3-1.5 reported plasmids.
Northern blot analysis
Northern blot was performed using nonisotopic digoxigenin (DIG) Northern starter kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol. Target fragments, mouse mimecan, and mouse glyceraldehye-3-phosphate dehydrogenase were cloned into PGEM-T Easy vector and confirmed by restriction enzyme digestion and sequence analysis. DIG-labeled probes were generated by transcription with SP6/T7 RNA polymerase using the DIG RNA labeling kit. Total RNA was isolated from mouse tissues by Trizol reagent (Invitrogen, Carlsbad, CA). The RNA content was estimated by spectrophotometry. Ten micrograms per lane were electrophoresed on a 1.2% agarose-formaldehyde denaturing gel and were transferred by capillary blotting to positively charged nylon membranes (Roche). The membrane was then baked at 80 C for 2 h. Hybridization was performed at 68 C with agitation overnight. The membrane was washed twice with 2x standard saline citrate (SSC) and 0.1% SDS for 5 min at room temperature and twice with 0.1x SSC and 0.1% SDS for 15 min at 68 C. The membrane was washed and blocked and then incubated with anti-DIG serum/alkaline phosphatase conjugate. CDP-Star (Roche) was used as the chemiluminescence substrate. Signals were visualized on x-ray film.
In situ hybridization
The RNA probes were labeled by using the DIG RNA labeling kit (SP6/T7; Roche). The pituitary gland from naturally aborted fetus and mouse was cut into serial frozen sections (5 µm). These sections were first fixed in 4% paraformaldehyde and digested in 1 µg/ml protein kinase buffer. After prehybridization, the sections were incubated with hybridization solution containing 0.5 ng/µl of probe in a humidified chamber overnight at 68 C. The posthybridization slides were washed twice with 2x SSCT (0.3 M sodium chloride, 30 mM sodium citrate, 0.1% Tween 20)/50% formamide at 68 C and once with 2x SSCT and 0.2x SSCT at room temperature and then incubated with anti-DIG-alkaline phosphatase Fab diluted 1:1000 in blocking solution. After being washed in MABT (0.1 M maleic acid, 0.15 M sodium chloride, 0.1 M Tris-base, 0.1% Tween 20, pH 7.5), they were incubated with staining buffer in a humidified chamber. To terminate the reaction, samples were rinsed several times with nuclease-free water and were visualized by light microscopy.
Immunohistochemical analysis
Sections of formalin-fixed, paraffin-embedded tissue specimens (5 µm thick) were rehydrated, and after antigen retrieval by microwave treatment, they were immunostained with polyclonal antibody of mimecan (1000-fold dilution), which was generated in our lab by immunizing rabbits with glutathione-S-transferase-mimecan fusion protein. Immunostaining was visualized with EnVision+ system (Dako, Carpinteria, CA), followed by nuclear counterstaining with Gill’s hematoxylin (Thermo-Shandon, Pittsburgh, PA). The preimmune rabbit serum (1000-fold dilution) was used as a negative control in adjacent sections.
Cell culture, transfections, and luciferase assays
SMMC-7721 cells (American Type Culture Collection, Manassas, VA), a cell type in which Pit-1 can be stably expressed by G418 screening, were seeded into 24-well plates, and maintained in DMEM, supplemented with 10% fetal bovine serum and antibiotics. At 80% confluence, cells were transiently transfected using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol.
For each well, 0.006 µg pRLO (Renilla luciferase internal control vector: SV40 promoter and enhancer to drive luciferase transcription; Promega) and 2 µl Lipofectamine were added in a transfection volume of 1 ml. The human mimecan promoter/luciferase reporter constructs, containing different lengths of the 5'-flanking region of the human mimecan gene and extending from –1350 to +1072 bp relative to the start site, were performed using the pGL3-basic firefly luciferase expression vector (Promega). The pGL3-2.4 construct contained 2.4 kb of the human mimecan promoter, including two Pit-1 REs. The pGL3-1.8 construct contained 1.8 kb of the human mimecan promoter in which one upstream Pit-1 RE sequence was deleted, so this plasmid contained only one Pit-1 RE. The shortest promoter construct was pGL3-1.5, in which two Pit-1 RE sequences were deleted. Transfections with the pGL3 basic plasmid were used for background determination, and transfections with the pGL3 control plasmid were used as positive controls. After 2 d, cells were harvested and luciferase activities were assessed using the Dual Luciferase assay kit (Promega). The firefly luciferase assays were then normalized with respect to Renilla luciferase activity. Identical transfections were performed in triplicate and repeated four times. The results are presented as the means with SE bars from four separate transfections performed.
EMSA
EMSA was performed using in vitro-translated protein Pit-1. Double-stranded oligonucleotide probes were used in this study. The oligonucleotides were as follows: probe 1, sense strand, 5'-taa taa aat ata tat cca tat ttg tta aat-3', and antisense strand, 5'-att taa caa ata tgg ata tat att tta tta-3'; probe 2, sense strand, 5'-aaa gct aat ttt tat gca tca gtt ttt ctt-3', and antisense strand, 5'-aag aaa aac tga tgc ata aaa att agc ttt-3'. These probes were 5' end-labeled with [-32P]dCTP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The labeled oligonucleotides were separated from the unincorporated nucleotides using a Probe Quant G-50 column (BioDirect, Taunton, MA). The 1-µl aliquots of the radiolabeled probe were incubated with 9 µl in vitro translation product for 30 min on ice in a 20-µl binding buffer (Promega). Specificity of protein binding to radiolabeled oligonucleotides was demonstrated by addition of a 10-fold excess of unlabeled competing oligonucleotide. After 20-min incubation at room temperature, the samples were resolved on a 4.5% polyacrylamide gel in 0.5x TBE (4.5 mM Tris-base, 4.5 mM boric acid, 0.1 mM EDTA) electrophoretic buffer at 100 V for 2 h at room temperature. After electrophoresis, the polyacrylamide gel was dried and autoradiographed.
Pituitary tumor samples
In total, 20 pituitary tumor samples were collected from January 1, 2002, to December 30, 2002, in Ruijin Hospital, Shanghai Second Medical University. These tumors consisted of three GH cell adenomas, four prolactin (PRL) cell adenomas, four ACTH cell adenomas, eight clinically nonfunctioning adenomas, and one multihormone tumor. Using immunohistochemical assay, the expression of mimecan protein in human pituitary was studied, and the results were compared with clinical, histological, and immunohistochemical features. In addition, expressions of GH, PRL, ACTH, FSH, LH, and TSH proteins were also studied and compared with that of mimecan protein.
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Results |
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We have established the gene expression profile of human pituitary, in which we have found high expression of mimecan in the pituitary (19). The present study was initiated to confirm that mimecan was expressed in the mouse pituitary gland by using Northern blot analysis. As shown in Fig. 1
, mimecan mRNA corresponding to a band of about 3.0 kb was present in a limited number of mouse tissues. It was most abundant in the lung and adrenal gland, although less abundant in the pituitary and heart. This result was confirmed by in situ hybridization. As shown in Fig. 2, mimecan mRNA was detected in the anterior lobe of the mouse pituitary with the antisense probe (Fig. 2A, arrow), although no signals were detected in this region with the sense probe (Fig. 2B). Using immunohistochemical assay, mimecan protein was also found to be localized in the human anterior pituitary (Fig. 2C, arrow). Taken together, these results showed that both mRNA and protein products of this gene are expressed in the pituitary.
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Interestingly, 18 binding sites for Pit-1 were predicted in bovine mimecan intron 1 sequence by bioinformatics (20). Using bioinformatics, two Pit-1 REs were identified in the 2.5-kb region of the human mimecan promoter, between 700 and 1000 bp upstream of the transcription initiation site. These data led us to further study whether the mimecan gene is regulated by Pit-1.
To test whether Pit-1 can bind to these two putative Pit-1 REs in vitro, EMSA was performed. The regions from nucleotides –203 to –163 bp and from –271 to –243 bp of the mimecan promoter, both of which contained the putative Pit-1 REs, were synthesized as probe 1 and probe 2, respectively. As shown in Fig. 3, purified Pit-1 proteins, which were prepared from an in vitro translation system with the pcDNA3-Pit1 expression plasmid, produced one major retarded band with probe 1 (lane 2) and probe 2 (lane 5), respectively, suggesting that Pit-1 might bind to the two putative Pit-1 REs of the human mimecan promoter. For cold competition assay, unlabeled oligonucleotides of probe 1 and probe 2 were added to the reactions as competitors. When 10-fold molar excess of unlabeled oligonucleotides of probe 1 and probe 2 (lanes 3 and 6) were added, retarded bands almost disappeared. No bands were detected for the nonspecific probe used as a negative control (lane 7). These results suggested that Pit-1 might be bound with the Pit-1 REs of the human mimecan promoter region in a sequence-specific manner.
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Next, the expression of the human mimecan and Pit-1 genes were screened by RT-PCR in many cell lines to identify the candidate cell for examining whether Pit-1 can regulate the transcriptional activity of human mimecan. Because the SMMC-7721 cells express the human mimecan gene at low level but do not express the Pit-1 gene, we could obtain a stable Pit-1 gene-expressing cell line by transfecting the Pit-1 gene. Then three mimecan promoter/luciferase reporter constructs containing different lengths of the 5'-flanking region of the gene (2.4, 1.8, and 1.5 kb) were generated (Fig. 4A), including two Pit-1 REs (pGL3-2.4), one Pit-1 RE (pGL3-1.8), or no Pit-1 RE (pGL3-1.5), respectively. These mimecan promoter/luciferase reporter plasmids were then transfected into SMMC-7721 cells with or without Pit-1 gene expression. As shown in Fig. 4B, luciferase activities of reporter plasmids, pGL3-2.4, pGL-1.8, and pGL-1.5 containing the two, one, or no Pit-1 gene binding site(s), respectively, were increased in the Pit-1 gene-expressing SMMC-7721 cells and in Pit-1 gene-deficient SMMC-7721 cells. This effect of the pGL3-2.4 plasmid was obviously greater in Pit-1 gene-expressing SMMC-7721 cells (22-fold) (Fig. 4B) than in Pit-1 gene-deficient SMMC-7721 cells (1.8-fold) (Fig. 4B). Moreover, deletion of the mimecan Pit-1 protein binding site impaired reporter gene activity. The activity of pGL3-1.8 mimecan reporter plasmid, which contained only the proximal Pit-1 protein binding site, was slightly higher in Pit-1 gene-expressing SMMC-7721 cells (4-fold) (Fig. 4B) than in Pit-1 gene-deficient SMMC-7721 cells (3-fold) (Fig. 4B). The activity of pGL3-1.5 mimecan reporter plasmid, without Pit-1 protein biding sites, is similar to the activity of pGL3-1.8 mimecan reporter plasmid (Fig. 4B). These results indicate that two Pit-1 protein binding sites in the regulatory region of the human mimecan gene are necessary for transcriptional activation by Pit-1 in transient transfections of SMMC-7721 cells. These results, together with the results of the EMSA experiment, indicate that transcriptional activation of the human mimecan promoter by Pit-1 is mediated by the sequences that bind Pit-1 in vitro.
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Because mimecan was detected in the normal human pituitary glands, we also investigated whether mimecan is expressed in different types of human pituitary tumors using immunohistochemical assay. Among 20 pituitary tumors, mimecan protein is expressed in 12 tumors (60%). Interestingly, six of seven cases of ACTH- or GH-secreting pituitary tumors express the mimecan protein, but only a portion of the PRL-, TSH-, FSH-, and LH-positive pituitary tumors express mimecan protein. Moreover, it was noticed that the expression of h-mimecan is found in all (four of four) of the clinical ACTH pituitary tumors, and in two of three clinical GH pituitary tumors. These data suggest that the ACTH or GH cell lineages secrete mimecan protein in human pituitary (Table 1 and Fig. 5). Unexpectedly, of eight clinically nonfunctioning pituitary tumors, four cases are positive for mimecan product.
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Discussion |
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and 2).
The mammalian pituitary gland plays a vital role in regulating the functions of the endocrine system. It regulates various endocrine organs by integrating the signals from the brain and the feedback effects of peripheral hormones to stimulate intermittent hormone release by a particular gland. The anterior pituitary synthesizes at least six hormones, which regulate growth, development, and function of the thyroid gland, adrenal cortex, gonads, and breasts. Up to now, it is known that six phenotypically distinct cell types, which secrete proopiomelanocortin (which is proteolytically cleaved to ACTH in corticotropes and MSH-
in melanotropes), GH in somatotropes, PRL in lactotropes, TSH in thyrotropes, and LH and FSH in gonadotropes, appear during anterior pituitary ontogeny in a characteristic order (21). The expression of the -glycoprotein subunit before the formation of the nascent pituitary, named Rathke’s pouch, heralds the onset of pituitary organogenesis (22). Primordial cell types are committed to the pituitary lineage through the induction of Lhx3/P-LIM expression, which may require the combinatorial actions of FGF8, BMP4, and Shh. Pitx2 is required for the expansion of these precursors within Rathke’s pouch, with Prophet of Pit-1 (Prop-1) required for the asymmetric ventral proliferation and determination of at least four ventral/intermediate cell types, namely somatotropes, lactotropes, thyrotropes, and gonadotropes (23). Pit-1 is subsequently required for the cell fate determination of three cell types (somatotropes, lactotropes, and thyrotropes), whereas GATA-2 is thought to be required for the thyrotrope and gonadotrope cell lineages, based on the presence or absence of Pit-1, respectively (24). The fact that targeted expression of Pit-1 ventrally in vivo is sufficient to convert gonadotropes to thyrotropes, whereas dorsal expression of GATA-2 is sufficient to convert all of the Pit-1-dependent cell types to gonadotropes through activation of factors such as SF1 (25), suggests that Pit-1 cell lineage and gonadotrope cell lineage probably arise from a common progenitor cell. However, differentiating from these four cell lineages, the melanotrope and corticotrope lineages, which arise from the dorsal precursor cells, are determined by a T-box factor, T-pit (26, 27) (Fig. 6). Our data have demonstrated that human mimecan protein was expressed in certain types of cells in the pituitary tumors, such as GH- and ACTH-secreting pituitary tumors. Although normal and hyperplastic pituitary tissues are polyclonal, abundant evidence suggests that pituitary adenomas are derived from monoclonal expansion of mutated somatic cells (28). Factors resulting in pituitary hyperplasia, including hypothalamic hormones, estrogens, and growth factors, likely facilitate a permissive intrapituitary milieu, potentiating genetic instability, cell mutation, and subsequent growth of already transformed adenomatous cells (28, 29). Thus, the fact that almost all GH- and ACTH-secreting pituitary tumors probably expressed mimecan protein suggested that the melanotrope and corticotrope lineage, Pit-1 cell lineage, and gonadotrope cell lineage probably originate from a common progenitor cell. However, the data are not conclusive because of the limited number of samples, and additional observation will be necessary to elucidate this possibility.
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There are six types of human pituitary tumors, corresponding to six types of pituitary tropic hormones plus a subtype for a nonfunctional pituitary tumor. Our observation may provide some insights into the origin of the cell types of some or the clinically nonfunctioning adenomas. The expression of human mimecan protein was positive in four of eight nonfunctioning pituitary tumors, which suggests that the mimecan, a protein not heretofore described in pituitary tissue, is synthesized in the subtype of pituitary nonfunctioning adenomas and may play a role in the morphological classification of pituitary nonfunctioning adenomas as a molecular marker.
In conclusion, this study demonstrates that mimecan is expressed in the mouse and human pituitary glands and that Pit-1 is able to enhance the transcriptional activity of the human mimecan gene. These observations also enhance the possibility that mimecan may be an unidentified pituitary secretory protein and that pituitary cells that secrete ACTH or GH may also secrete mimecan.
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Acknowledgments |
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Footnotes |
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First Published Online September 27, 2005
1 S.-M.H., F.L., and H.-M.Y. contributed equally to this work. 
Abbreviations: BMP, Bone morphogenetic protein; DIG, digoxigenin; IRF-1, interferon regulatory factor-1; Pit-1, pituitary transcription factor-1; PRL, prolactin; RE, response element; SLRP, small leucine-rich proteoglycan; SSC, standard saline citrate.
Received February 14, 2005.
Accepted September 15, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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regulation of the human mimecan promoter. Mol Vis 9:277–287[Medline]
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