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Endocrine-Related Cancer 14 (3) 875-886    DOI: 10.1677/ERC-07-0036
Copyright © 2007 by the Society for Endocrinology.
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The Mia/Cd-rap gene expression is downregulated by the high-mobility group A proteins in mouse pituitary adenomas

 Ivana De Martino1, Rosa Visone1, Dario Palmieri1, Paolo Cappabianca2, Paolo Chieffi3, Floriana Forzati1, Antonio Barbieri4, Mogens Kruhoffer5, Gaetano Lombardi6, Alfredo Fusco1,7 and Monica Fedele1

1 Dipartimento di Biologia e Patologia Cellulare e Molecolare and Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, University of Naples ‘Federico II’, via Pansini 5, 80131 Naples, Italy
2 Dipartimento di Scienze Neurologiche, Divisione di Neurochirurgia, University of Naples ‘Federico II’, Naples, Italy
3 Dipartimento di Medicina Sperimentale, II University of Naples, Naples, Italy
4 Istituto dei Tumori di Napoli Fondazione ‘G Pascale’, Naples, Italy
5 Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark
6 Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, University of Naples ‘Federico II’, Naples, Italy
7 NOGEC (Naples Oncogenomic Center), CEINGE Biotecnologie Avanzate and SEMM, European School of Molecular Medicine, Naples, Italy

(Correspondence should be addressed to A Fusco; Email: afusco{at}napoli.com)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The high-mobility group A (HMGA) family of proteins orchestrates the assembly of nucleoprotein structures playing important roles in gene transcription, recombination, and chromatin structure through a complex network of protein–DNA and protein–protein interactions. Recently, we have generated transgenic mice carrying wild type or truncated HMGA2 genes under the transcriptional control of the cytomegalovirus promoter. These mice developed pituitary adenomas secreting prolactin and GH mainly due to an increased E2F1 activity, directly consequent to the HMGA2 overexpression. To identify other genes involved in the process of pituitary tumorigenesis induced by the HMGA2 gene, in this study we have analyzed the gene expression profile of three HMGA2-pituitary adenomas in comparison with a pool of ten normal pituitary glands from control mice, using the Affymetrix MG MU11K oligonucleotide array representing ~13 000 unique genes. We have identified 82 transcripts that increased and 72 transcripts that decreased at least four-fold in all the mice pituitary adenomas analyzed compared with normal pituitary glands. Among these genes, we focused our attention on the Mia/Cd-rap gene, whose expression was essentially suppressed in all of the pituitary adenomas tested by the microarray. We demonstrated that the HMGA proteins directly bind to the promoter of the Mia/Cd-rap gene and are able to downregulate its expression. In order to understand a possible role of Mia/Cd-rap in pituitary cell growth, we performed a colony assay in GH3 and GH4 cells. Interestingly, Mia/Cd-rap expression inhibits their proliferation, suggesting a potential tumor suppressor role of Mia/Cd-rap in pituitary cells.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The high-mobility group A (HMGA) family consists of three proteins: HMGA1a and HMGA1b that result from alternative splicing of the same gene, i.e. HMGA1 (Johnson et al. 1989), and HMGA2, which is encoded by a different gene (Manfioletti et al. 1991). They are small nuclear proteins that bind to the minor groove of DNA through highly cationic regions called ‘AT-hooks’ (Reeves 2001). The HMGA proteins function as an architectural transcription factor: they do not independently regulate gene transcription, but modulate gene expression through the formation of stereospecific complexes on the promoter/enhancer regions of genes by direct interaction with other transcription factors and through substrate interactions that bend, unwind, or distort the structure of DNA (Thanos & Maniatis 1995). Both the genes have a critical role in the development, during which they are abundantly expressed. In fact, the generation of HMGA2-knockout mice resulted in a pygmy phenotype associated with a drastic reduction of the fat tissue (Zhou et al. 1995), whereas HMGA1 null mice showed cardiac hypertrophy and type 2 diabetes (Foti et al. 2005, Fedele et al. 2006a).

Rearrangements of the HMGA2 gene have been frequently found in human benign tumors, mainly of mesenchymal origin, such as lipomas, lung hamartomas, and uterine leiomyomas (Ashar et al. 1995, Schoenmakers et al. 1995). Both the HMGA genes have a critical role in the process of carcinogenesis because they are over-expressed in most human malignant neoplasias (Melillo et al. 2001) and the blockage of their expression has been shown to prevent thyroid cell transformation and lead malignant cells to death (Scala et al. 2000, Berlingieri et al. 2002). Moreover, both HMGA1 and HMGA2 behave as classical oncogenes in focus assays on mouse and rat fibroblasts (Fedele et al. 1998, Wood et al. 2000). The generation of transgenic mice overexpressing either the HMGA1 or the HMGA2 gene confirmed their oncogenicity also in vivo. In fact, both the HMGA1 and HMGA2 transgenic mice develop growth hormone/prolactin (GH/PRL)-secreting pituitary adenomas and T/NK lymphomas (Baldassarre et al. 2001, Fedele et al. 2002, Fedele et al. 2005).

Consistently with the development of pituitary adenomas in HMGA2 transgenic mice, HMGA2 gene amplification and overexpression have been shown in a large set of human prolactinomas supporting a critical role of HMGA2 in this human neoplasia (Finelli et al. 2002). The mechanism by which HMGA2 is involved in pituitary tumorigenesis is on the ability of the HMGA2 to interfere with the pRB/E2F1 pathway. In fact, we have recently shown that HMGA2 interacts with retinoblastoma protein (pRB) and induces an increased E2F1 activity in pituitary adenomas by displacing histone deacetylase (HDAC1) from the pRB/E2F1 complex and resulting in E2F1 acetylation (Fedele et al. 2006b). The suppression of pituitary tumorigenesis by mating HMGA2TG and E2F1–/– mice demonstrates a critical role for the HMGA2-mediated E2F1 activation in the onset of these tumors in transgenic mice, and likely in human prolactinomas.

Although the E2F1 activation might represent a major point in the generation of pituitary adenomas in transgenic mice, we cannot exclude the fact that other complementary mechanisms may be envisaged for the role of HMGA2 in pituitary tumorigenesis. In fact, also in the E2F1 minus background, the HMGA2 mice develop a certain number of pituitary neoplasias, even though with a lower frequency and a minor phenotype. Thus, the aim of the present work has been to find out other molecular changes that might contribute to the development of the HMGA2-induced pituitary tumors. Therefore, we analyzed the expression profile of three pituitary adenomas developed by HMGA2 transgenic mice in comparison with a pool of normal pituitary glands from wild-type animals. We screened an array in which ~13 000 were represented, and we identified 82 transcripts that increased and 72 that decreased with a greater than or equal to four-fold change in pituitary adenomas versus normal ones. These results were validated by semiquantitative reverse transcription (RT)-PCR performed on pituitary tumors originating from different HMGA2 transgenic mice. Then, we focused our attention on the Mia/Cd-rap gene, whose expression was drastically downregulated in HMGA2-induced pituitary adenomas. Mia/Cd-rap is a small, secreted protein that is expressed normally at the onset of chondrogenesis (Dietz & Sandell 1996). Interestingly, it is secreted by malignant melanoma cells and elicits growth inhibition of melanoma cells in vitro (Blesch et al. 1994).

Here we report that the HMGA proteins are able to bind to the promoter of the Mia/Cd-rap gene both in vitro and in vivo indicating a direct role of HMGA proteins in the regulation of the transcription of the Mia/Cd-rap gene. Functional studies by luciferase assays confirmed the critical role of the HMGA proteins in the downregulation of the Mia/Cd-rap promoter. To understand the relevance of Mia/Cd-rap downregulation in pituitary adenoma cell growth, we expressed Mia/Cd-rap in GH3 and GH4 cells and performed colony assays. Consistently with a putative tumor suppressor role for Mia/Cd-rap in pituitary cells, we found that its expression causes growth inhibition.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
RNA extraction

Pituitary glands adenomas from wild type and HMGA2 mice (Fedele et al. 2002, 2006b) were snap-frozen in liquid nitrogen and stored at –80 °C until use. Total RNAs were extracted from tissues and cell lines using TRI REAGENT (Molecular Research Center Inc., Cincinnati, OH, USA) solution, according to manufacturer’s instructions. The integrity of the RNA was assessed by denaturing agarose gel electrophoresis (virtual presence of sharp 28S and 18S bands) and spectrophotometry.

Microarray analysis

The Affymetrix standard protocol has been described extensively elsewhere (Affymetrix GeneChip). Briefly, cRNA was prepared from 8 µg total RNA, hybridized to MG MU11K Affymetrix oligonucleotide arrays (containing about 13 000 murine transcripts), scanned, and analyzed according to Affymetrix (Santa Clara, CA, USA) protocols. Scanned image files were visually inspected for artifacts and normalized by using GENECHIP 3.3 software (Affymetrix). The individual gene expression levels for each of the three pituitary adenomas arrays were divided by the expression level in the normal pituitary tissue. Thus, the data were presented as relative to the expression in normal pituitary tissue. The fold change values, indicating the relative change in the expression levels between mutated and wild-type samples, were used to identify genes differentially expressed between these conditions.

Cluster analysis by Multiexperiment Viewer (MeV)

Microarray data have been elaborated by the MeV system to get gene expression signature of the samples analyzed. The MeV is a system of cluster analysis for genome wide expression data from DNA microarray hybridization that uses standard statistical algorithms to arrange genes according to similarity in the pattern of gene expression. In our analysis, we used a four-fold difference in expression level between normal and tumoral samples.

Semiquantitative RT-PCR

RNAs were treated with DNaseI (Invitrogen) and reverse transcribed using random exonucleotides and MuLV reverse transcriptase (Perkin–Elmer, Waltham, MA, USA). To ensure that RNA samples were not contaminated with DNA, negative controls were obtained by performing PCR on samples that were not reverse transcribed, but otherwise identically processed. For semiquantitative PCR, reactions were optimized for the number of cycles to ensure product intensity within the linear phase of amplification. The PCR products were separated on a 2% agarose gel, stained with ethidium bromide, and scanned using a Typhoon 9200 scanner. Digitized data were analyzed using Imagequant (Molecular Dynamics, Sunnyvale, CA, USA). Primers sequences and different annealing conditions are available as supplemental data (Supplemental Table 1Go, which can be viewed online at http://erc.endocrinology-journals.org/supplemental/).


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  		Table 1 Genes differentially expressed with a greater than or equal to four-fold change in high-mobility group A 2 (HMGA2)-induced pituitary adenomas versus normal pituitary gland
 
Cell cultures and transfections

All cell lines, except for {alpha}T3-1 (kindly provided by Dr P Mellon, University of California, San Diego, CA, USA), were purchased from ATCC. They were all cultured in DMEM containing 10% fetal bovine serum (HyClone, Logan, UT, USA) and 50 µg/ml gentamicin (Life Technologies Inc.) in a humidified atmosphere of 95% air and 5% CO2. B16F0 are murine melanoma cells; At T20 and {alpha}T3-1 are murine pituitary adenoma cells secreting adrenocorticotrophin (ACTH) and gonadotropic hormones, respectively; RC-4B/C are rat pituitary adenoma cells secreting GH, follicle-stimulating hormone, luteinizing hormone, gonadotrophin-releasing hormone, ACTH, and thyrotrophin (TSHb); GH1, GH3, and GH4 are rat pituitary adenoma cells secreting PRL and GH.

Transfections were carried out by using Lipofectamine 2000, according to manufacturer’s instructions.

Plasmids

The 5' flanking region of the mouse Mia/Cd-rap gene spanning nucleic acid residues –1396 to –1 with respect to the ATG protein start codon was amplified by PCR and inserted into the promoterless luciferase plasmid pGL3-basic (Promega) to obtain the MIA-luc pGL3 plasmid. The human Mia/Cd-Rap expression plasmid, pCMV6-XL4/Mia, was commercially available (TC116021 – OriGene Technologies, Rockville, MD, USA). pBABE-puro had been already described (Monaco et al. 2001).

Luciferase and colony assays

For the Luciferase assay, a total of 2 x 105 B16F0 cells were seeded into each well of six-well plates and were transiently transfected with 1 µg MIA-luc pGL3 and with the indicate amounts of pCEFLHa-HMGA1 (Melillo et al. 2001) and pCEFLHa-HMGA2 (Fedele et al. 2006b), together with 0.5 µg Renilla and various amounts of the pCEFLHa plasmid to keep the total DNA concentration constant. Transfection efficiencies were normalized by using Renilla luciferase expression assayed with the dual luciferase system (Promega). All transfection experiments were repeated at least three times.

For the colony assay, GH3 and GH4 cells were seeded at a density of 2.5 x 106 per 10 mm dish. Two days after, the cells were transfected with 10 µg pCMV6-XL4 plus 2 µg pBabe-puro, or 10 µg pCMV6-XL4/Mia plus 2 µg pBabe-puro. After about 15 days, the cells were stained with 500 mg/ml crystal violet in 20% methanol, and the resulting colonies were counted.

Protein extraction and western blot

Tissues and cell culture were lysed in buffer 1% NP40, 1 mmol/l EDTA, 50 mmol/l Tris–HCl (pH 7.5), and 150 mmol/l NaCl, supplemented with complete protease inhibitors mixture (Roche Diagnostic Corp). Total proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% nonfat milk and incubated with antibody against MIA (A-20 Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-17047). Bound antibody was detected by the appropriate secondary antibody and revealed with an enhanced chemiluminescence system (Amersham-Pharmacia Biotech).

Electrophoretic mobility shift assay (EMSA)

Protein/DNA-binding was determined by EMSA, as previously described (Battista et al. 1995). Briefly, 5–20 ng of recombinant protein were incubated in the presence of a 32P-end-labeled double-strand oligonucleotide (specific activity, 8000–20 000 c.p.m./fmol). Spanning from base –1130 to –1100 of the mouse Mia/Cd-rap promoter region (5'-AAACCCTGAAATAAATCTTTTTTTCCCCTT-3'). The DNA–protein complexes were resolved on 6% non-denaturing acrylamide gels and visualized by exposure to autoradiographic films.

Chromatin immunoprecipitation (ChIP)

ChIP was carried out with an acetyl-histone H3 immunoprecipitation assay kit (Upstate Biotechnology, Charlottesville, VA, USA) according to manufacture’s instruction. Approximately, 3 x 107 cells of the NIH3T3 cell line were grown on 75 cm2 dishes and cross-linked by the addition of formaldehyde (to 1% final concentration) to the attached cells. Cross-linking was allowed to proceed at room temperature for 5 min and was terminated with glycine (final concentration, 0.125 mol/l). The cells were collected and lysed in buffer containing 5 mmol/l piperazine-N, N'-bis[2-ethanesulfonic acid] (PIPES); (pH 8.0), 85 mmol/l KCl, 0.5% NP40, and protease inhibitors(1 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), on ice for 10 min. Nuclei were pelleted by centrifugation at 2300 g for 5 min at 4 °C and resuspended in buffer containing 50 mmol/l Tris–Cl (pH 8.1), 10 mmol/l EDTA, 1% SDS, the same protease inhibitors, and incubated on ice for 10 min. Chromatin was sonicated on ice to an average length of about 400 bp with a Branson sonicator model 250. Samples were centrifuged at 16 000 g for 10 min at 4 °C. Chromatin was pre-cleared with protein A Sepharose (blocked previously with 1 mg/ml BSA) at 4 °C for 2 h. Pre-cleared chromatin of each sample was incubated with 2 µg antibody anti-HA (sc-7392, Santa Cruz Biotechnology) at 4 °C overnight. An aliquot of wild-type sample was incubated also with anti-IgG antibody. Next, 60 µl a 50% slurry of blocked protein A Sepharose was added and immune complexes were recovered. The supernatants were saved as ‘input.’ Immunoprecipitates were washed twice with 2 mmol/l EDTA, 50 mmol/l Tris–Cl (pH 8.0) buffer and four times with 100 mmol/l Tris–Cl (pH 8.0), 500 mmol/l LiCl, 1% NP40, and 1% deoxycholicacid buffer. The antibody-bound chromatin was eluted from the beads with 200 µl elution buffer (50 mmol/l NaHCO3, 1% SDS). The samples were incubated at 67 °C for 5 h in thepresence of 10 µg RNase and NaCl to a final concentration of 0.3 mol/l to reverse formaldehyde cross-links. The samples were then precipitated with ethanol at –20 °C overnight. The pellets were resuspended in 10 mmol/l Tris (pH 8)–1 mM EDTA and treated with proteinase K to a final concentration of 0.5 mg/ml at 45 °C for 1 h. DNA was extracted with phenol/chloroform/isoamylalcohol, ethanol precipitated, and resuspended in water. Input DNA and immunoprecipitated DNAs were analyzed by PCR for the presence of Mia/Cd-rap promoter sequence. The PCR were performed with AmpliTaq gold DNA polymerase (Perkin–Elmer). The primers used to amplify the sequence of the Mia/Cd-rap promoter were 5'-TTGCTGGTGCATGCCTTA-3' (forward) and 5'-TCTTAACCGCTGAGCCATCT-3' (reverse). The PCR products were resolved on a 2% agarose gel, stained with ethidium bromide, and scanned using a Typhoon 9200 scanner.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Gene expression profile analysis

RNAs were extracted from a pool of normal pituitary glands and from three pituitary adenomas developed in three different HMGA2 transgenic mice, and were hybridized to one MG Affymetrix MU11K-A oligonucleotide array containing about 13 000 transcripts. The number of transcripts that increased or decreased in all pituitary adenomas versus normal gland is shown in the Fig. 1Go. Of the 13.059 transcripts represented on the array, 1560 had a one- to three-fold, 290 had a three- to four-fold, 154 had a four- to ten-fold, and 11 had a greater than ten-fold change. We examined the 154 transcripts that had a fold change ≥4 in all HMGA2-induced pituitary adenomas versus normal pituitary gland. Among these transcripts, 82 were increased and 72 were decreased, including 108 known genes, 30 expression sequence tags and 16 unknown genes. The relative fold changes of these genes, grouped according to their biological function, are shown in Table 1Go.


Figure 1
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  		Figure 1 Gene expression profile of pituitary adenomas of HMGA2 transgenic mice. The expression profile of three HMGA2-induced pituitary adenomas was compared with that of normal pituitary gland of wild-type mice. Transcripts are grouped according to fold change.

 
A natural basis for organizing gene expression data is to group together genes with similar patterns of expression. Using the MeV, a free Java application which is used to compare datasets from microarray expression experiments (Eisen et al. 1998), we clustered the gene expression profile of HMGA2-induced pituitary adenomas in comparison with the normal gland. The results, illustrated in Fig. 2Go, clearly show the similar pattern of expression of several genes in all pituitary adenomas with respect to the normal pituitary gland.


Figure 2
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  		Figure 2 Clustered display of gene expression in three pituitary adenomas developed in three HMGA2 transgenic mice when compared with normal pituitary gland using the MEV program. A four-fold difference in expression level between adenomas and normal samples was used. The green and red colors show downregulated and upregulated genes respectively. Each gene is represented by a single row of colored boxes; each adenoma is represented by a single column.

 
Validation of microarray data

To validate the results obtained by microarray analysis, we evaluated the expression of about 100 transcripts, whose expression differed of a fold change > 4 or ≤4 in all the three pituitary adenomas compared with normal glands. To this aim, we performed semiquantitative RT-PCRs in normal glands and HMGA2-induced pituitary adenomas derived from mice different from those used for the Gene Chip microarray. For all of these genes, we confirmed the differential expression associated with the pituitary tumors. The results of some representative RT-PCR analyses are shown in Fig. 3AGo. Among these differentially expressed genes, there are some upregulated (e.g. Cyclin B2, ANKT and noggin) and other downregulated (e.g. Rib-1, CamK2 and Mia/Cd-rap) in pituitary tumors with respect to the normal gland. Most of the genes analyzed show the same regulation in their expression also in pituitary adenomas developed in transgenic mice overexpressing the HMGA1 gene (Fedele et al. 2005). Some of these representative results are shown in the Fig. 3BGo.


Figure 3
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  		Figure 3 Validation of microarray data by semiquantitative RT-PCR. (A) Representative RT-PCRs on a panel of three HMGA2-induced pituitary adenomas and a pool of wild-type pituitary glands. (B) Representative RT-PCRs on a panel of two HMGA1-induced pituitary adenomas and a pool of wild-type pituitary glands. (C) Some of the genes regulated in HMGA-induced pituitary adenomas were assayed by semiquantitative RT-PCR on a panel of seven cell lines originating by rat and mouse pituitary adenomas of different histotype in comparison with a pool of wild-type mouse pituitary glands. Amplification of the ß-actin gene has been evaluated as a control of the RNA amount used.

 
Subsequently, we analyzed by RT-PCR the expression of these genes also in murine and rat cell lines derived by pituitary adenomas of different histotype in comparison with normal mouse or rat pituitary gland (Fig. 3CGo and data not shown). Some genes, such as Mia/Cd-rap and CamK2, as for the pituitary adenomas of HMGA mice, are downregulated in all the cell lines, whereas other genes, such as FKBP65 or reelin, change their expression depending on the cellular histotype.

HMGA proteins regulate Mia/Cd-rap expression

We concentrated our attention on the Mia/Cd-rap gene for two main reasons: i) it was the most downregulated gene in all of the HMGA2-induced pituitary adenomas analyzed and ii) it has been already associated with tumor development, even though it is upregulated in different human neoplasias such as chondrosarcoma, melanoma, and breast cancer (Blesch et al. 1994, Chansky et al. 1998).

Mia/Cd-rap protein is downregulated in mouse pituitary adenomas
To further validate the microarray data obtained for the Mia/Cd-rap gene, we also analyzed the Mia/Cd-rap protein expression in tissue extracts of pituitaries and pituitary adenomas from control and transgenic mice (either HMGA1 or HMGA2) respectively. As shown in Fig. 4AGo, normal pituitary from control mice show high levels of Mia/Cd-rap protein expression, whereas it was completely lost in pituitary adenomas from both HMGA1 and HMGA2 transgenic mice.


Figure 4
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  		Figure 4 HMGA proteins regulate the murine Mia/Cd-rap promoter. (A) Mia/Cd-rap protein expression in pituitary adenomas and normal pituitary glands from HMGA (both HMGA1 and HMGA2) transgenic mice and wild-type animals respectively. (B) EMSA performed with a radiolabeled oligonucleotide spanning from base –1130 to –1100 of the mouse Mia/Cd-rap promoter region, and incubated with 5 ng (lanes 5) and 20 ng (lanes 6) of the recombinant HMGA1-His protein or with 5 ng (lane 1) and 20 ng (lanes 2) of the recombinant HMGA2-His protein. Where indicated, a 100-fold molar excess of unlabelled MIApr oligonucleotide was added as a specific competitor. (C) Chromatin immunoprecipitation assay performed on NIH3T3 cells transfected with pCEFLHA, pCEFLHa-HMGA1, or pCEFLHa-HMGA2. The recovered DNA was used as a template for PCR with primers that specifically amplify the murine Mia/Cd-rap promoter. Input indicates PCR products with chromosomal DNA without immunoprecipitation. As an immunoprecipitation control, IgG was used. (D) Luciferase activity (fold of activation) of the Mia/Cd-rap promoter in the B16F0 murine melanoma cell line. Where indicated 0.5 and 1 µg of either HMGA1 or HMGA2 expression vectors were cotransfected with the Mialuc plasmid.

 
HMGA proteins bind to the Mia/Cd-rap promoter
To investigate whether the HMGA proteins are directly involved in Mia/Cd-rap transcriptional regulation, we evaluated the HMGA-binding activity to the Mia/Cd-rap promoter in vitro using oligonucleotides spanning from base –1130 to –1100 of the mouse Mia/Cd-rap promoter region (Bosserhoff et al. 1997), including AT-rich putative HMGA-binding sites. As shown in Fig. 4BGo, lanes 1–2, increasing amounts (5 and 20 ng) of the recombinant HMGA2 protein were capable of binding the 32P-end-labeled double-strand oligonucleotide in EMSA. The binding specificity was demonstrated by competition experiments showing loss of binding with the addition of 100-fold molar excess of the specific unlabeled oligonucleotide (lane 3). We performed the same experiment with a recombinant HMGA1 protein, and also in this case a specific binding with the Mia/Cd-rap promoter (Fig. 4BGo, lanes 5–7) was detected.

To verify that HMGA proteins are able to bind to Mia/Cd-rap promoter in vivo, we performed experiments of ChIP in the NIH3T3 cell line transiently transfected with either the HA-HMGA2 or HA-HMGA1 expression plasmids. Chromatin prepared as described under Materials and methods was immunoprecipitated with anti-HA or normal rabbit IgG antibody. The results, shown in Fig. 4CGo, confirmed that both HMGA2 and HMGA1 proteins bind to the promoter of Mia/Cd-rap gene. In fact, the Mia/Cd-rap promoter region was amplified from the DNA recovered with anti-HA antibody in HA-HMGA1- and HA-HMGA2- but not in mock-transfected cells. Moreover, no amplification was observed in the samples immunoprecipitated with a specific rabbit IgG.

HMGA proteins regulate the Mia/Cd-rap promoter activity
To define the functional consequences of the interaction between HMGA proteins and Mia/Cd-rap promoter, we co-transfected the B16F0 cell line, in which Mia/Cd-rap protein is endogenously expressed, with a construct expressing the luciferase reporter gene under the control of the mouse Mia/Cd-rap promoter region –1396 to +1 (mMIApromluc) and increasing the amounts of an HMGA2 (or HMGA1) expression vector. As shown in Fig. 4DGo, the overexpression of HMGA2 (or HMGA1) resulted in a decreased activity of the Mia/Cd-rap promoter in a dose-dependent manner. Interestingly, the HMGA1 protein showed a significantly higher inhibitory effect on the Mia/Cd-rap promoter activity in comparison with the HMGA2 protein (80% with 1 µg HMGA1 versus 55% with 1 µg HMGA2).

Mia/Cd-rap expression inhibits growth of pituitary adenoma cells

To investigate the functional role of Mia/Cd-rap in pituitary cell growth, we performed colony assay experiments by transfecting Mia/Cd-rap in pituitary adenoma GH3 and GH4 cells. After puromycin selection, the number and growth of the colonies obtained by transfection with the Mia expression plasmid decreased dramatically compared with empty vector in both cell lines (Fig. 5Go). These results show that Mia/Cd-rap inhibits the cell growth of rat pituitary adenoma cells, suggesting it as a negative regulator of pituitary cell proliferation.


Figure 5
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  		Figure 5 Effect of Mia/Cd-rap expression on pituitary adenoma cell proliferation. Colony forming assay experiment performed on GH3 (black bars) and GH4 (gray bars) cells transfected with a vector expressing Mia/Cd-rap. As a control the empty vector (mock) was used. The reported results are the mean of three experiments and error bars show S.D.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Pituitary tumors constitute about 15% of intracranial neoplasms and are mostly benign with slow growth. Most pituitary neoplasms secrete hormone gene products, leading to disturbed endocrine functions. About two-thirds of pituitary tumors express and secrete pituitary hormones. Among these, prolactinomas are the most common lesions, representing about 50% of all the clinically diagnosed pituitary adenomas. The genesis of pituitary tumors remains still largely unknown, and several factors, such as gene mutations, locally produced growth factors, and hypothalamic dysregulation, play a critical role in the transformation of pituitary cells. A crucial role for the HMGA2 gene in the process leading to the generation of human pituitary adenomas was first suggested by the phenotype of transgenic mice overexpressing a truncated or a wild-type HMGA2 gene under the transcriptional control of the strong and ubiquitous promoter of the cytomegalovirus. Most of these mice (85% of females and 40% of males), in fact, developed pituitary adenomas secreting PRL and GH (Fedele et al. 2002). After these findings, we investigated the role of the HMGA2 gene in human prolactinomas. Interestingly, cytogenetic analysis of prolactinomas revealed not only an increased dosage of the HMGA2 gene, which resulted from multiple mechanisms, mainly simple chromosome gain (trisomy or tetrasomy of chromosome 12), but also over-representation of the HMGA2 gene through der (12) chromosomes (Finelli et al. 2002). The strong correlation between the phenotype of the HMGA2 transgenic mice and the HMGA2 overexpression in human prolactinomas suggested that this animal model might represent a unique tool for studying the molecular mechanisms underlying the generation of pituitary adenomas induced by the HMGA2 over-expression. In fact, these mice allowed us to demonstrate that the mechanism of the HMGA2-induced pituitary adenoma development is based on the increased E2F1 activity (Fedele et al. 2006b). Since other additional mechanisms can be envisaged on the base of a minimal residual tumoral phenotype showed by some HMGA2 transgenic mice lacking a functional E2F1 gene (Fedele et al. 2006c), in the present study we have analyzed the gene expression profile of three HMGA2-pituitary adenomas in comparison with a pool of ten normal pituitary glands in order to identify other genes involved in the process of pituitary tumorigenesis induced by the HMGA2 gene. The results of our analysis led to the identification of 82 transcripts that increased and 72 transcripts that decreased at least fourfold in all mice pituitary adenomas analyzed when compared with normal pituitary gland. It is noteworthy that among the overexpressed genes, we found galectin-3. This is consistent with previous reports showing a role for the galectin-3 protein in pituitary cell proliferation and tumor progression (Riss et al. 2003). Here we focused on the Mia/Cd-rap gene because its expression was essentially suppressed in all of the mouse pituitary adenomas analyzed and, even though it has been already associated with a neoplastic phenotype (Blesch et al. 1994, Chansky et al. 1998), it has been never described in pituitary tumorigenesis so far. We demonstrated, by EMSA and ChIP, that both HMGA1 and HMGA2 proteins directly bind to the promoter of this gene and downregulate its expression in transcription assays. It is noteworthy to observe that even though both the HMGA proteins are able to regulate the Mia/Cd-rap gene, this effect is more pronounced with HMGA2 than with HMGA1. Finally, by colony assays in GH3 and GH4 cells we were able to demonstrate that re-expression of Mia in this pituitary adenoma cell line dramatically reduces their proliferative potential, indicating a potential tumor suppressor role for this gene in pituitary tumors.

The protein Mia/Cd-rap is mainly expressed in cartilage during embryogenesis (Dietz & Sandell 1996), and is related to cellular motility, metastasis, and modulation of immune responses in melanoma cells. In fact, it has been shown that Mia/Cd-rap interacts with components of the extracellular matrix, such as laminin and fibronectin, suggesting that it may have a function in regulating detachment of melanoma and possibly other cells from the extracellular matrix, which is an important step in metastasis (Bosserhoff et al. 1997).

Therefore, the previous published data about the Mia/Cd-rap gene appear in contrast with those reported here which suggest a tumor suppressor function of this gene rather than an oncogenic function. It is reasonable to retain that, as it occurs for other proteins, such as HMGA1 (Martinez Hoyos et al. 2004), the Mia/Cd-rap protein function may depend on the cellular context. Interestingly, a recent study proves that HMG1, another member of the HMG family, is an important factor in MIA regulation and melanoma progression. In fact, a sequence-specific DNA motif in the highly conserved region of the Mia/Cd-rap promoter is recognized by HMG1 (Golob et al. 2000). Besides, recent data showed that HMG1 is upregulated in malignant melanoma cell lines compared with normal human embryonal melanocytes (NHEM) normal skin cell line and that it plays a pivotal role in Mia/Cd-rap transcriptional activation (Poser et al. 2003). Consistently, the promoter sequence of Mia/Cd-rap contains many potential regulatory domains including an AT-rich domain, which are known as DNA-binding sites for the HMGA proteins (Bosserhoff et al. 1997).

In conclusion, in this study we have identified Mia/Cd-rap as a gene directly downregulated by the HMGA proteins in HMGA-induced pituitary adenomas. Moreover, we showed that Mia/Cd-rap expression is inversely correlated with the proliferative potential of pituitary adenoma cells, thereby suggesting a relevant role of its downregulation in the generation of pituitary adenomas.


   Acknowledgements
 
This work was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC) to Alfredo Fusco and Regione Campania to Monica Fedele. We thank the Associazione Partenopea per le Ricerche Oncologiche (APRO) for its support. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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