TSH-induced gene expression involves regulation of self-renewal and differentiation-related genes in human bone marrow-derived mesenchymal stem cells

in Journal of Endocrinology
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  • 1 Immunology Research Center, Department of Immunology, Department of Hematology, Endocrinology Department, Gazi University, 06500 Ankara, Turkey

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Bone marrow-derived mesenchymal stem cells are pluripotent cells that are capable of differentiating into a variety of cell types including neuronal cells, osteoblasts, chondrocytes, myocytes, and adipocytes. Despite recent advances in stem cell biology, neuroendocrine relations, particularly TSH interactions remain elusive. In this study, we investigated expression and biological consequence of TSH receptor (TSHR) interactions in mesenchymal stem cells of cultured human bone marrow. To the best of our knowledge, we demonstrated for the first time that human bone marrow-derived mesenchymal stem cells expressed a functional thyrotropin receptor that was capable of transducing signals through cAMP. We extended this study to explore possible pathways that could be associated directly or indirectly with the TSHR function in mesenchymal stem cells. Expression of 80 genes was studied by real-time PCR array profiles. Our investigation indicated involvements of interactions between TSH and its receptor in novel regulatory pathways, which could be the important mediators of self-renewal, maintenance, development, and differentiation in bone marrow-derived mesenchymal stem cells. TSH enhanced differentiation to the chondrogenic cell lineage; however, further work is required to determine whether osteoblastic differentiation is also promoted. Our results presented in this study have opened an era of regulatory events associated with novel neuroendocrine interactions of hypothalamic–pituitary axis in mesenchymal stem cell biology and differentiation.

Abstract

Bone marrow-derived mesenchymal stem cells are pluripotent cells that are capable of differentiating into a variety of cell types including neuronal cells, osteoblasts, chondrocytes, myocytes, and adipocytes. Despite recent advances in stem cell biology, neuroendocrine relations, particularly TSH interactions remain elusive. In this study, we investigated expression and biological consequence of TSH receptor (TSHR) interactions in mesenchymal stem cells of cultured human bone marrow. To the best of our knowledge, we demonstrated for the first time that human bone marrow-derived mesenchymal stem cells expressed a functional thyrotropin receptor that was capable of transducing signals through cAMP. We extended this study to explore possible pathways that could be associated directly or indirectly with the TSHR function in mesenchymal stem cells. Expression of 80 genes was studied by real-time PCR array profiles. Our investigation indicated involvements of interactions between TSH and its receptor in novel regulatory pathways, which could be the important mediators of self-renewal, maintenance, development, and differentiation in bone marrow-derived mesenchymal stem cells. TSH enhanced differentiation to the chondrogenic cell lineage; however, further work is required to determine whether osteoblastic differentiation is also promoted. Our results presented in this study have opened an era of regulatory events associated with novel neuroendocrine interactions of hypothalamic–pituitary axis in mesenchymal stem cell biology and differentiation.

Introduction

The TSH receptor or the thyrotropin receptor (TSHR) is usually expressed by thyroid cells. It is a G protein-coupled receptor and signals through cAMP and inositol triphosphate (IP3) pathways (Kursawe & Paschke 2000, Davies et al. 2002, Szkudlinski et al. 2002). Stimulation of the TSHR via TSH on thyroid cells leads to production of the thyroid hormones (THs), tri-iodothyronine (T3) and thyroxine (T4) (Toccafondi et al. 1982). The TSHR expression by other tissue cells has also been reported (Haraguchi et al. 1996, Hoermann 1996, Dutton et al. 1997, Bell et al. 2000, Agretti et al. 2002, 2005, Tsai et al. 2004, Ellerhorst et al. 2006). However, exact function of its expression in extrathyroidal tissues has not been clarified.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) have been characterized by many properties so far. They are pluripotent cells that are capable of differentiating into a variety of mesenchymal tissues (Pittenger et al. 1999, Kemp et al. 2005, Kolf et al. 2007, Abdallah & Kassem 2008). Despite these progressions, neuroendocrine interactions in many aspects of mesenchymal stem cell maintenance and differentiation are still largely unknown. For example, whether hBMSCs express the TSHR is totally unknown. Certainly, biological consequences of interactions between the TSH and its receptor in maintenance and differentiation of hBMSCs also remain to be elucidated.

In this study, we aimed to investigate the expression and biological function(s) of the TSHR in hBMSCs. TSH-induced gene expression was explored particularly.

Materials and Methods

Isolation and maintenance of mesenchymal stem cells

hBMSCs were isolated by plastic adherence according to a previously published method (François et al. 2006) from iliac crest aspirates of volunteer donors. Volunteer donors of allogeneic bone marrow transplant recipients signed an informed consent, and the isolation protocol was approved by the local ethics committee of Gazi University Medical School. Isolated mesenchymal stem cells were maintained by culturing them in DMEM supplemented with 10% fetal bovine serum, 4 mM l-glutamine, penicillin–streptomycin, non-essential amino acid solution, and pyruvate (all from Gibco-Invitrogen) at 37 °C and 5% CO2. The medium was replaced every 3 days.

Immunofluorescence and immunohistochemistry

hBMSCs were grown on chamber slides (Nunc, Roskilde, Denmark) for staining to visualize TSHR. For immunohistochemistry, a commercially available staining kit was used according to the manufacturer's recommendations (Dako, Via Real Carpinteria, CA, USA). Briefly, cells were fixed with ice-cold methanol (or in some cases with 4% paraformaldehyde) for 20 min at room temperature and then stained with antihuman TSHR antibodies (Mouse IgG1 and IgG2a, ABR, Rockford, IL, USA) at room temperature for 2 h in the presence of Hoechst 33342 Fluorescent stain (Invitrogen). After washing extensively, slides were treated with anti-mouse IgG-FITC (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h. Confocal laser scanning microscopy (Leica, Wetzlar, Germany) was used to visualize slides.

Antihuman COL2A1 and antihuman COL9A1 antibodies (Abcam, Cambridge, MA, USA) were used to detect protein expression for human COL2A1 and COL9A1. Fixed cells were stained with primary antibodies at room temperature for 2 h. Slides were washed extensively and incubated with an anti-rabbit secondary antibody conjugated with HRP. Stained cells were visualized using a Leica microscope with software called ‘The Leica Application Suite’ (Leica, DM4000).

FACS analysis

Adherent cells were trypsinized and washed twice. Cells were stained with monoclonal antibodies for 30 min at 4 °C, washed twice, and fixed with 2% paraformaldehyde before use. Anti-CD13, CD14, anti-CD29, anti-CD34, anti-CD45, anti-CD73, anti-CD105 antibodies, and PE- or FITC-labeled isotype-matched control antibodies were used (eBioscience, San Diego, CA, USA). Analyses were performed using a Cytomics FC500 flow cytometer with CXP software (Beckman Coulter, Brea, CA, USA).

Immunoprecipitation and western blotting

Immunoprecipitation and western blot experiments were conducted using a previously published protocol (Bagriacik & Klein 2000). Briefly, cell lysate was incubated overnight at 4 °C with an antihuman TSHR antibody of mouse (ABR) and immunoprecipitated with agarose-conjugated protein A (Sigma). Ten percent polyacrylamide ready-made gels (Thermo Scientific, Rockford, IL, USA) were used for electrophoresis. Immunoblots were treated with another clone of antihuman TSHR primary antibody (ABR) overnight and then with an anti-mouse-HRP secondary antibody (Jackson Immunoresearch) for 2 h at room temperature. Enhanced chemiluminescence detection system was used for protein detection (Pierce-Thermo Scientific, Rockford, IL, USA).

Assay for cAMP

A commercially available colorimetric assay kit (Assay Design, Ann Arbor, MI, USA) was used to analyze the cAMP change following TSH stimulation. Briefly, hBMSCs were starved for 24 h in serum-free culture medium and then stimulated by the recombinant TSH at logarithmically increasing concentrations. Each concentration was tested in triplicate wells. Measurements were performed at 450 nm using a multi-mode microplate reader (Synergy HT, BioTek, Winooski, VT, USA).

Genomic DNA-free total RNA isolation

Total RNA was isolated using a genomic DNA-free total RNA isolation kit (Qiagen). Cells were lysed by the lysis solution provided in the kit content and treated with RNase-free DNAase as instructed before passing through RNA isolation columns. Extracted RNA was quantified and stored at −86 °C until use.

Real-time RT-PCR arrays

Gene expression was assessed using a RT2 Profiler PCR Array kit obtained from SuperArray Bioscience Corporation (Frederick, MD, USA). The Human Stem Cell RT2 Profiler PCR Array including the expression of 80 genes of 14 different pathways and markers related to the identification, growth, and differentiation of stem cells was chosen. The kit also contained the stem cell-specific markers as stem cell differentiation markers and genes in signaling pathways important for stem cell maintenance. One microgram total RNA per sample was converted into cDNA in reverse transcriptase (RT) reaction. Real-time PCR was performed using a Light Cycler 480 (Roche Applied Science). Amplification continued for 40 cycles. Results were calculated as fold increase values of gene expression as instructed by the kit supplier. At least a twofold increase or a twofold decrease in a particular gene expression over control values was considered as a statistically significant (P<0.05) change.

Alcian blue staining

Alcian blue (Sigma) was prepared using 3% glacial acetic acid. Fixed cells were stained with 0.1% alcian blue for 30 min. Cells were washed extensively and visualized.

Statistical analyses

Statistical data were calculated by Student's t-test or two-way ANOVA as indicated with the level of significance set at P<0.05 or P<0.01. Relative changes as fold change values in gene expression were calculated by the formula using ΔΔCt method (threshold).

Results

Bone marrow-derived cells exhibited the characteristics of mesenchymal stem cells

We isolated hBMSCs by plate attachment method to study the TSHR expression and its consequences in mesenchymal stem cells. Confluently grown cells in cultures were typical mesenchymal stem cells as described by other investigators (Pittenger et al. 1999, Kolf et al. 2007, Abdallah & Kassem 2008). They were adherent monolayer of spindle-shaped cells having a fibroblastic appearance. We molecularly characterized cultured MSCs by flow cytometry (FACS) to make sure that the isolated cells from human bone marrow were mesenchymal stem cells. For this reason, expression of CD13, CD14, CD29, CD34, CD73, and CD105 was determined.

There is a growing list of evidence about age-associated decline in MSC-specific marker expression. To find culture stability and purity, we also determined expression of those MSC markers during early, middle, and late passages of cultured cells. All of the cultured cells at the 5th passage representing early passages expressed CD13, CD29, CD73, and CD105 at high levels while they lacked the expression of CD14 and CD34 (Fig. 1). They did not express CD45 either (data not shown). Decline in the frequency of MSC-specific marker expressing cells begun after the 10th passage (data not shown). At the 12th passage, as the mid-passage, only 30–35% of cultured cells were positive for CD13, CD29, CD73, and CD105 (Fig. 1). None of the cultured cells expressed any of those markers at the 20th passage as the late passage (Fig. 1). Based on these data, we concluded that our isolates of bone marrow-derived cells were indeed mesenchymal stem cells, and they maintained surface expression of those mesenchymal stem cell markers at high levels until the 10th passage (data not shown). Therefore, we used the cells from the 5–6th passages for TSHR expression and gene expression studies.

Figure 1
Figure 1

FACS analysis of specific markers for hBMSCs. Cells were stained with anti-CD13, anti-CD14, anti-CD29, anti-CD34, anti-CD73, and anti-CD105 antibodies. PE- or FITC-labeled isotype-matched antibodies were used as control. Cells from early (the 5th passage), middle (the 12th passage), and late passages (the 20th passage) were tested. Numbers at the corners represent percent values of positive cells for indicated markers.

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0404

TSHR expression by mesenchymal stem cells

TSHR expression was explored by three powerful methods for protein detection and visualization. Western blot analysis was performed using hBMSCs isolated from two different donors. Two bands of different size appeared on the blot. The larger band was about 100 kDa and the smaller one was about 65 kDa (Fig. 2A). The results were confirmed by repeats of four independent experiments. TSHR was also visualized by using immunofluorescence of confocal laser microscopy. Apparently, hBMSCs in culture expressed a homogeneous expression pattern of TSHR at relatively high density (Fig. 2B). We also tested TSHR expression by flow cytometry. MSCs from different passages expressed the TSHR (Fig. 2C). These data clearly showed that TSHR with a homogeneous pattern was constitutively expressed on hBMSCs.

Figure 2
Figure 2

Determination of TSHR expression. TSHR expression on the cell surface of hMSCs was determined by three different methods. (A) Western blot: lane 1, TSHR in human thyroid cells; lane 2, TSHR in hBMSCs from the first donor; and lane 3, TSHR in hBMSCs from the second donor. (B) In fluorescence confocal microscopy studies, cells were stained with the primary antihuman TSHR antibody and FITC-conjugated secondary Ab plus Hoechst to stain the nuclei. Inverted Leica DM4000 with 4-laser Leica confocal microscope system (Leica GE) and software, Wetzlar, Germany. (C) TSHR expression was also assessed by flow cytometry.

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0404

TSHR signaling through cAMP

The next question we asked was whether the TSHR expressed on the hBMSCs was a functional receptor. Since TSHR is a G protein-coupled receptor, we measured cAMP levels following stimulation by TSH. To stimulate the receptor under in vitro conditions in cell cultures, we used a commercially available recombinant human TSH (thyrogen), which contained both α and β chains of the hormone. cAMP production increased in a dose-dependent manner (Fig. 3) upon stimulation and reached the peak level at a concentration of 10−8 M recombinant TSH. Forskolin treatment served as a positive control for induction of cAMP production.

Figure 3
Figure 3

Determination of cAMP in TSH-treated hBMSCs. An ELISA-based cAMP assay was used to measure cAMP increase followed by TSH treatment. Forskolin served as a positive control. Assay was performed in triple wells per sample. The mean value and the s.d.s were calculated.

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0404

Induction of specific gene expression by TSH

To study biological consequences of TSHR expression in the life of hBMSCs, we investigated TSH-induced expression of genes that were related to the stem cell-specific markers, stem cell differentiation markers, and signaling pathways important for stem cell maintenance. Quantitative gene expression profiles were studied by real-time RT-PCR technology using highly purified DNA-free RNA samples from stimulated cells by the recombinant TSH.

We studied several stem cell-specific markers that were categorized under various groups of functional gene families such as cell adhesion molecules, chromosome and chromatin modulators, cell cycle regulators, cytokines and growth factors, and genes regulating cell–cell communication. As some of those genes would have more than one function and can be categorized in overlapping groups, we included them in only one family while evaluating our results. For example, APC gene is categorized among both cell adhesion molecules and cell cycle regulators. The gene expression results for APC gene was demonstrated only under the group of cell adhesion molecules in Table 1.

Table 1

Induction of gene expression for stem cell specific markers

Fold change
GenbankFunction10−7 Ma10−8 Ma
Genes
Cell adhesion molecules
 APCNM_000038Adenomatous polyposis coli+1.02−1.17
 BGLAPNM_199173Bone gamma-carboxyglutamate (gla) protein (osteocalcin)+1.18−1.25
 CD44NM_000610CD44 molecule (Indian blood group)+1.0−1.33
 CDH1NM_004360Cadherin 1, type 1, E-cadherin (epithelial)+1.2+1.71
 CDH2NM_001792Cadherin 2, type 1, N-cadherin (neuronal)+1.12+1.04
 CTNNA1NM_001903Catenin (cadherin-associated protein), alpha 1, 102 kDa+1.03+1.01
 CXCL12NM_000609Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)−1.36−1.56
 NCAM1NM_000615Neural cell adhesion molecule 1−1.19+1.05
Chromosome and chromatin modulators
 KAT2ANM_021078General control of amino-acid synthesis 5-like 2 (yeast)−1.39−1.14
 HDAC2NM_001527Histone deacetylase 2+1.38+1.67
 MYST1NM_032188MYST histone acetyltransferase 1−1.04+1.02
 MYST2NM_007067MYST histone acetyltransferase 2−1.37−1.38
 RB1NM_000321Retinoblastoma 1 (including osteosarcoma)−1.09−1.05
 TERTNM_198253TERT Telomerase reverse transcriptase+2.35*+5.80*
Cell cycle regulators
 AXIN1NM_003502Axin 1−1.54−3.34*
 CCNA2NM_001237Cyclin A2+1.11+1.07
 CCND1NM_053056Cyclin D1−1.30+1.43
 CCND2NM_001759Cyclin D2−1.08+1.13
 CCNE1NM_001238Cyclin E1−1.30−1.33
 CDK1NM_001786Cell division cycle 2, G1 to S and G2 to M+1.07+1.10
 CDC42NM_001791Cell division cycle 42 (GTP binding protein, 25 kDa)−1.24−1.27
 EP300NM_001429E1A binding protein p300−1.39−2.43*
 MYCNM_002467V-myc myelocytomatosis viral oncogene homolog (avian)−1.19−2.95*
 PARD6ANM_016948Par-6 partitioning defective 6 homolog alpha (Caenorhabditis elegans)−1.23−1.29
Cytokines and growth factors
 BMP1NM_006129Bone morphogenetic protein 1−1.41−1.68
 BMP2NM_001200Bone morphogenetic protein 2−1.02+1.45
 BMP3NM_001201Bone morphogenetic protein 3 (osteogenic)+2.90*+6.31*
 CXCL12NM_000609Chemokine (C-X-C motif) ligand 12−1.38−1.56
 FGF2NM_002006Fibroblast growth factor 2 (basic)−1.43−1.44
 FGF3NM_005247Fibroblast growth factor 3+6.31*+18.35*
 FGF4NM_002007Fibroblast growth factor 4+11.37*+31.51*
 GDF2NM_016204Growth differentiation factor 2+3.06*+9.76*
 GDF3NM_020634Growth differentiation factor 3+4.78*+18.73*
 IGF1NM_000618IGF1 (somatomedin C)−1.08+1.23
 JAG1NM_000214Jagged 1 (Alagille syndrome)−1.96−2.14*
Genes regulating cell–cell communication
 DHHNM_021044Desert hedgehog homolog (Drosophila)+1.5+3.06*
 DLL1NM_005618Delta-like 1 (Drosophila)+1.5+2.54*
 GJA1NM_000165Gap junction protein, alpha 1, 43 kDa−1.19−1.18
 GJB1NM_000166Gap junction protein, beta 1, 32 kDa+2.54*+4.78*
 GJB2NM_004004Gap junction protein, beta 2, 26 kDa+1.54+3.0

*P<0.05.

TSH concentrations used to stimulate hBMSCs.

Treatment of hBMSCs with the recombinant TSH resulted in upregulation or downregulation of gene expression not only in a selective way but also in a dose-dependent manner, i.e. 10−8 M TSH affected gene expression more than 10−7 M TSH. We observed a moderate increase in TERT, BMP3, and GDF2 genes (Table 1). On the other hand, we found sharp increases in the expression of FGF3, FGF4, and GDF3 (Table 1). ATP-binding cassette sub-family G member 2 (ABCG2), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), aldehyde dehydrogenase 2 family (mitochondrial) (ALDH2), and fibroblast growth factor receptor 1 (FGFR1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)) are among the metabolic markers for stem cells. TSH induced both ABCG2 and ALDH1A1 (Fig. 4A). Increase in the expression of ABCG2 was moderate. However, gene expression of ALDH1A1 increased almost 19 times upon TSH challenge at 10−8 M in comparison with the untreated controls.

Figure 4
Figure 4

Regulation of gene expression of hBMSCs. (A) Metabolic markers: ATP-binding cassette sub-family G (white) member 2 (ABCG2), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), aldehyde dehydrogenase 2 family (mitochondrial) (ALDH2), fibroblast growth factor receptor 1 (FGFR1). (B) Self-renewal markers of hBMSCs: heat-shock 70 kDa protein 9 (HSPA9), MYST histone acetyltransferase 1 (MYST1), MYST histone acetyltransferase 2 (MYST2), neurogenin 2 (NEUROG2), sex-determining region Y (SRY)-box 1 (SOX1), sex-determining region Y (SRY)-box 2 (SOX2). (C) Gene expression for signaling pathways related to stem cell maintenance such as WNT pathway: adenosine deaminase RNA-specific (ADAR), adenomatous polyposis coli (APC), axin 1 (AXIN1), beta-transducin repeat containing (BTRC), cyclin D1 (CCND1), frequently rearranged in advanced T-cell lymphomas (FRAT1), frizzled homolog 1 (Drosophila) (FZD1), V-myc myelocytomatosis viral oncogene homolog (avian) (MYC), peroxisome proliferator-activated receptor delta (PPARD), wingless-type MMTV integration site family, member 1 (WNT1). (D) Notch pathway: delta-like 1 (Drosophila) (DLL1), delta-like 3 (Drosophila) (DLL3), Deltex homolog 1 (Drosophila) (DTX1), Deltex homolog 2 (Drosophila) (DTX2), Dishevelled, dsh homolog 1 (Drosophila) (DVL1), E1A binding protein p300 (EP300), GCN5 general control of amino-acid synthesis 5-like 2 (yeast) (GCN5L2 or KAT2A), histone deacetylase 2 (HDAC2), Jagged 1 (JAG1), Notch homolog 1, translocation-associated (NOTCH1), Notch homolog 2 (NOTCH2), Numb homolog (NUMB) *(P<0.05).

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0404

Self-renewal is an essential characteristic of embryonic stem cells. We investigated whether self-renewal-related markers were affected by TSH in hBMSCs. Sex-determining region Y (SRY)-box 1 (SOX1), sex-determining region Y (SRY)-box 2 (SOX2), neurogenin 2 (NEUROG2), MYST1, MYST2, and heat-shock 70 kDa protein 9 (HSPA9) genes were tested. TSH selectively and significantly augmented NEUROG2 and SOX2 expression, whereas TSH decreased or did not affect the remaining genes (Fig. 4B). The effect of the TSH was again dose dependent. SOX2 expression increased by ∼8- and 21-fold at 10−7 and 10−8 M TSH application, respectively, while the increase in NEUROG2 expression was relatively lower and moderate.

We also found that the gene expressions of the two important signaling pathways related to stem cell maintenance were also affected by TSH. Ten genes from WNT pathway and 12 genes from Notch pathway were studied. Remarkably, only WNT1 within the WNT pathway was upregulated by almost 11 times at 10−8 M TSH (Fig. 4C). On the other hand, we observed increases in several members of the Notch pathways. For example, transcription factors such as DTX1 and DLL3 increased by ten times and 16 times, respectively (Fig. 4D).

Other genes that were affected by TSH

To find the induction of gene expression that was influenced by the presence of the recombinant TSH in hBMSCs, we tested genes related to stem cell differentiation and to the signaling pathways being important for stem cell maintenance. Mesenchymal cell lineage markers, embryonic cell lineage markers, and neuronal cell lineage markers were included among the stem cell differentiation markers. TSH treatment changed expression of markers such as COL2A1 and COL9A1. In particular, COL2A1 expression increased almost 26-fold whereas COL9A1 increased about eightfold (Table 2). Interestingly, TSH treatment abrogated alkaline phosphatase (ALPI) expression by more than 200-fold decline while it did not affect PPARG expression significantly. Decrease in aggrecan gene expression was important as well (Table 2). These data suggested that TSH might contribute to the regulation of differentiation processes by which mesenchymal stem cells differentiate into chondrocyte-like cells in cultures. However, one may argue that increase in gene expression at mRNA level may not reflect protein expression. Therefore, we first studied protein expression for COL2A1 and COL9A1. Assessment of protein expression was performed using immunohistochemistry. Our findings showed that COL2A1 protein expression elevated in TSH-treated cells (Fig. 5B). However, COL9A1 protein expression was less than COL2A1 expression (Fig. 5A). In supporting the protein expression studies, we treated cells with TSH for 21 days in order to follow differentiation process. Treated MSCs stained alcian blue positive (Fig. 5C). These data suggested that MSCs had capacity to differentiate into chondrocyte-like cells in the presence of TSH.

Table 2

Induction of gene expression for stem cell differentiation markers

Fold change
GenbankFunction10−7 Ma10−8 Ma
Genes
Mesenchymal cell lineage markers
 ACANNM_001135Aggrecan−1.45−3.53*
 ALPINM_001631Alkaline phosphatase, intestinal−226.28*−103.39*
 BGLAPNM_199173Bone gamma-carboxyglutamate (gla) protein (osteocalcin)+1.18−1.25
 COL1A1NM_000088Collagen, type I, alpha 1−1.61−1.59
 COL2A1NM_001844Collagen, type II, alpha 1+12.71*+25.59*
 COL9A1NM_001851Collagen, type IX, alpha 1+4.22*+7.45*
 PPARGNM_015869Peroxisome proliferator-activated receptor gamma+1.32+1.96
Neural cell lineage markers
 NCAM1NM_000615Neural cell adhesion molecule 1−1.19+1.05
 OPRS1NM_005866Opioid receptor, sigma 1+1.37−1.04
 S100BNM_006272S100 calcium binding protein B+2.90*+3.45*
 TUBB3NM_006086Tubulin, beta 3−1.11−1.45
Embryonic cell lineage markers
 ACTC1NM_005159Actin, alpha, cardiac muscle 1+7.66*+13.81*
 ASCL2NM_005170Achaete–scute complex homolog 2 (Drosophila)+4.88*+9.11*
 FOXA2NM_021784Forkhead box A2+1.12+2.21*
 PDX1NM_000209Pancreatic and duodenal homeobox 1+4.22*+7.40*
 ISL1NM_002202ISL LIM homeobox 1+1.67+2.82*
 KRT15NM_002275Keratin 15+2.08*+3.47*
 MSX1NM_002448Msh homeobox 1+1.10−1.04
 MYOD1NM_002478Myogenic differentiation 1+2.05*+7.98*
 TNM_003181T, brachyury homolog (mouse)+2.59*+5.49*

*P<0.05.

TSH concentrations used to stimulate hBMSCs.

Figure 5
Figure 5

Chondrogenic-like cell differentiation and protein expression of COL2A1 and COL9A1. (A) Immunohistochemistry for COL9A1 detection. (B) Immunohistochemistry for COL2A1 detection. (C) Alcian blue staining of differentiated cells by TSH stimulation.

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0404

Among the embryonic stem cell lineage markers tested, significant upregulations in ACTC1, ASCL2, PDX1, and T expression occurred. ASCL2 elevated by ninefold while ACTC1 increased by 13-fold in comparison with the control group, which was not treated with the recombinant TSH (Table 2). Elevations in PDX1 and T were also significant but relatively lower (Table 2).

Discussion

In this study, we showed that hBMSCs expressed a functional TSHR that was able to signal through cAMP upon stimulation by a recombinant TSH. More importantly, we explored TSH-induced gene expression in hBMSCs. Our results indicated that TSH affected expression patterns of many genes involved in various developmental and differentiation pathways in mesenchymal stem cell life and provided further support for the existence of regulatory roles of hypothalamic–pituitary–thyroid axis in the developmental stages of hBMSCs.

It is very well known that stimulation of TSHR by TSH results eventually in production of THs, T3 and T4 in thyroid cells (Toccafondi et al. 1982, Szkudlinski et al. 2002). However, we also know that TSHR can be expressed by extrathyroidal tissues or cells (Haraguchi et al. 1996, Hoermann 1996, Dutton et al. 1997, Bell et al. 2000, Agretti et al. 2002, 2005, Tsai et al. 2004, Ellerhorst et al. 2006). For example, kidney cells were shown to express a functional TSHR that transmitted signals through cAMP (Sellitti et al. 2000). Additionally, dendritic cells and a few subsets of T cells express functional TSHR that is capable of signaling via both cAMP and JAK2 (Whetsell et al. 1999, Bagriacik & Klein 2000). These previously accumulated data indicate that TSHR expressed in extrathyroidal tissues probably mediates differential functions. Increased phagocytosis of fluorescent-labeled particles and elevated cytokine secretions are a few examples for variable consequences of TSH-induced gene expression in dendritic cells (Bagriacik & Klein 2000). In fact, our results in this study showed that stimulation via TSHR induced a variety of gene expressions, at least at mRNA level. Particularly, genes involved in self-renewal and maintenance were affected by the presence of TSH.

Several studies showed that SOX2 and OCT4 gene expressions were required for self-renewal process in embryonic stem cells (Niwa et al. 2000, Ivanova et al. 2006). Additionally, forced expression of SOX2 was related to maintenance of expansion and differentiation capabilities of hBMSCs (Go et al. 2008) implying that maintenance of expansion governed by SOX2 would be regulated by TSH–TSHR interactions. Also, we know that SOX2 is in the regulation of FGF4 expression in embryonic stem cells (Johnson et al. 1998, Kamachi et al. 2000, Gao et al. 2009). Our results showed that TSH upregulated expressions of both FGF4 and FGF3 significantly. Therefore, we speculated that TSH would affect FGF4 expression probably by increasing SOX2 expression through an indirect way. However, it remains to be investigated.

COL2A1 and COL9A1 are among the lineage markers for mesenchymal stem cells. COL9A1 gene encodes one of the four α chains found in type IX collagen. COL2A1 is the gene encoding for the α chain of type II collagen. Type IX collagen is closely associated with type II collagen in cartilage components (Wu et al. 1992, Eyre et al. 2004). Members of the SOX family have been found to be responsible for the transcriptional regulation of type II and IX collagen genes including COL2A1 and COL9A1 (Bell et al. 1997, Stokes et al. 2001). Particularly, SOX9 played an important role in COL9A1 gene expression (Zhang et al. 2003). Therefore, one can argue that via members of SOX family, TSH may be involved in the regulation of collagen synthesis during cartilage generation. In fact, Miura et al. (2002) showed that THs enhanced cartilage differentiation in the growth plate in organ-cultured mouse tibias. By doing so, longitudinal bone growth was stimulated. They also found that T3 exposure increased expression of type II collagen gene in ATDC5 cells, a mouse chondrogenic cell line. Their findings about THs having functional effects in chondrocyte differentiation would be strong support for our study. However, our argument about induction of chondrogenic lineage activities in hBMSCs is a consequence of a direct TSH–TSHR interaction rather than a TH–TH receptor interaction.

MSCs that were incubated in the presence of TSH for 21 days stained alcian blue positive. Alcian blue, a phthalocyanine, reacts with proteoglycans such as chondroitin residue, hyaluronic residue, and sulfated residue. Therefore, it can be used for detection of chondrogenic cell differentiation (Quintarelli et al. 1964, Scott et al. 1964, Cowman et al. 1984). Significant increases in alcian blue positivity and COL2A1 expression in TSH-treated cells were indications of chondrogenic-like cell differentiation. Taken all together, these data suggested that TSH–TSHR interaction enhanced commitment of hMSCs to differentiate toward chondrogenic-like cells. However, further studies are also necessary to understand whether TSH–TSHR interaction may promote osteogenic differentiation as well.

It is believed that WNT, FGF, Notch, Hedgehog, and TGFh/BMP signaling network is involved in the maintenance of tissue homeostasis not only by regulating self-renewal of normal stem cells but also by regulating proliferation or differentiation of progenitor cells as well (Katoh & Katoh 2007). We found that WNT1 gene of the WNT pathway was upregulated significantly by TSH. Also TSH induced gene expression of important members of the Notch signaling pathway. In particular, inductions in expression of DLL3 and DTX1 were significantly high. Current literature has no satisfactory information concerning a distinct correlation between those genes and any selective function. In fact, we observed selective and important upregulations among members of WNT, FGF, Notch, and BMP signaling network. Many other genes, which were known to be important in developmental events in stem cell's life, were affected by the recombinant TSH treatment. Therefore, one can argue that TSH indeed may contribute to maintenance and the regulations of self-renewal and tissue homeostasis of hBMSCs during their proliferation and differentiation process. As a support for our speculation, Kim et al. (2007) found that TSH increased expression of WNT1 gene in FRTL5 cells, a rat thyroid cell line.

The constitutive expression pattern of the TSHR suggested that the TSHR might be required to play an essential role in self-renewal, maintenance, and differentiation processes as a mediator of activating a variety of pathways. In establishing functions associated with the expression of the TSHR, we proposed a primitive model of signaling events (Fig. 6) based on the data we obtained in the study. This new model would serve as a base to establish further molecular pathways as a part of neuroendocrine interactions of the hypothalamic–pituitary axis.

Figure 6
Figure 6

A model for associated functions with TSHR expression in hBMSCs. The TSHR is a G protein-coupled receptor. When TSH binds to its receptor, subunits of the G protein activate some biochemical pathways as shown in the figure. As a consequence of these activations, expressions of some transcription factors or some growth factors are regulated in processes of self-renewal, differentiation, and maintenance. TSH receptor (TSHR), G protein s (Gs), G protein q (Gq), protein kinase A (PKA), phosphatidyl inositol 4,5-bisphosphate (PIP2), inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG), protein kinase C (PKC), and Janus kinase 2 (JAK2).

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0404

Taken all together, experimental system in this study has proven that hBMSCs expressed a functional TSHR. This is the first original report about the expression of TSHR by hBMSCs and also indicates the induction of various genes that are involved in regulations of critical pathways in the mesenchymal stem cell life. Our findings strongly suggested that TSH and TSHR interaction was related to induction of several genes regulating self-renewal, cellular maintenance, and differentiation processes such as cartilage differentiation from hBMSCs. However, currently we do not know how the TSH is involved in orchestrating a hierarchy of regulated transcription factors that are critical for self-renewal and differentiation pathways. The precise sequential mechanism as the consequences of interactions between the TSHR and TSH is under investigation in our laboratories. To support the results of our in vitro experimental system, we plan to design in vivo studies in the future.

In conclusion, this study has provided novel information regarding the regulatory role of neuroendocrine interactions of the hypothalamic–pituitary axis in human mesenchymal stem cell biology, self-renewal, and differentiation. TSH–TSHR interaction would be one particular mechanism of triggering those regulatory events. Future studies should clarify the remaining questions in establishing the biological importance of the TSHR expression by hBMSCs.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported by a governmental grant from the State Planning Organization in Turkey (DPT 2003K120470-37 and 2009K120670).

Acknowledgements

The authors thank Prof. Dr Sevim Ercan for her kind help in the confocal microscopy experiments. They also thank Prof. Dr John R Klein from The Dental Branch of The University of Texas at Houston for his critical review of the manuscript and for his kind advice.

References

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    • Search Google Scholar
    • Export Citation
  • Agretti P, Chiovato L, De Marco G, Marcocci C, Mazzi B, Sellari-Franceschini S, Vitti P, Pinchera A & Tonacchera M 2002 Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues. European Journal of Endocrinology 147 733739. doi:10.1530/eje.0.1470733.

    • Search Google Scholar
    • Export Citation
  • Agretti P, De Marco G, De Servi M, Marcocci C, Vitti P, Pinchera A & Tonacchera M 2005 Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation. European Journal of Endocrinology 152 777784. doi:10.1530/eje.1.01900.

    • Search Google Scholar
    • Export Citation
  • Bagriacik EU & Klein JR 2000 The thyrotropin (thyroid-stimulating hormone) receptor is expressed on murine dendritic cells and on a subset of CD45RB high lymph node T cells: functional role for thyroid-stimulating hormone during immune activation. Journal of Immunology 164 61586165.

    • Search Google Scholar
    • Export Citation
  • Bell DM, Leung KK, Wheatley SC, Ng LJ, Zhou S, Ling KW, Sham MH, Koopman P, Tam PP & Cheah KS 1997 Sox9 directly regulates the type-II collagen gene. Nature Genetics 16 174178. doi:10.1038/ng0697-174.

    • Search Google Scholar
    • Export Citation
  • Bell A, Gagnon A, Grunder L, Parikh SJ, Smith TJ & Sorisky A 2000 Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts. American Journal of Physiology. Cell Physiology 279 C335C340.

    • Search Google Scholar
    • Export Citation
  • Cowman MK, Slahetka MF, Hittner DM, Kim J, Forino M & Gadelrab G 1984 Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides. Biochemical Journal 221 707716.

    • Search Google Scholar
    • Export Citation
  • Davies T, Marians R & Latif R 2002 The TSH receptor reveals itself. Journal of Clinical Investigation 110 161164.

  • Dutton CM, Joba W, Spitzweg C, Heufelder AE & Bahn RS 1997 Thyrotropin receptor expression in adrenal, kidney, and thymus. Thyroid 7 879884. doi:10.1089/thy.1997.7.879.

    • Search Google Scholar
    • Export Citation
  • Ellerhorst JA, Sendi-Naderi A, Johnson MK, Cooke CP, Dang SM & Diwan AH 2006 Human melanoma cells express functional receptors for thyroid-stimulating hormone. Endocrine-Related Cancer 13 12691277. doi:10.1677/erc.1.01239.

    • Search Google Scholar
    • Export Citation
  • Eyre DR, Pietka T, Weis MA & Wu JJ 2004 Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage. Journal of Biological Chemistry 279 25682573. doi:10.1074/jbc.M311653200.

    • Search Google Scholar
    • Export Citation
  • François S, Bensidhoum M, Mouiseddine M, Mazurier C, Allenet B, Semont A, Frick J, Saché A, Bouchet S & Thierry D et al. 2006 Local irradiation not only induces homing of human mesenchymal stem cells at exposed sites but promotes their widespread engraftment to multiple organs: a study of their quantitative distribution after irradiation damage. Stem Cells 24 10201029. doi:10.1634/stemcells.2005-0260.

    • Search Google Scholar
    • Export Citation
  • Gao F, Kwon SW, Zhao Y & Jin Y 2009 PARP1 poly (ADP-ribosylates Sox2 to control Sox2 protein levels and FGF4 expression during embryonic stem cell differentiation. Journal of Biological Chemistry 284 2226322273. doi:10.1074/jbc.M109.033118.

    • Search Google Scholar
    • Export Citation
  • Go MJ, Takenaka C & Ohgushi H 2008 Forced expression of Sox2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities. Experimental Cell Research 314 11471154. doi:10.1016/j.yexcr.2007.11.021.

    • Search Google Scholar
    • Export Citation
  • Haraguchi K, Shimura H, Lin L, Saito T, Endo T & Onaya T 1996 Functional expression of thyrotropin receptor in differentiated 3T3-L1 cells: a possible model cell line of extrathyroidal expression of thyrotropin receptor. Biochemical and Biophysical Research Communications 223 193198. doi:10.1006/bbrc.1996.0868.

    • Search Google Scholar
    • Export Citation
  • Hoermann R 1996 Stimulation of thyroidal and extrathyroidal thyrotropin receptors. Experimental and Clinical Endocrinology and Diabetes 104 (Suppl 4) 8891. doi:10.1055/s-0029-1211710.

    • Search Google Scholar
    • Export Citation
  • Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun Y & Lemischka IR 2006 Dissecting self-renewal in stem cells with RNA interference. Nature 442 533538. doi:10.1038/nature04915.

    • Search Google Scholar
    • Export Citation
  • Johnson LR, Lamb KA, Gao Q, Nowling TK & Rizzino A 1998 Role of the transcription factor Sox-2 in the expression of the FGF-4 gene in embryonal carcinoma cells. Molecular Reproduction and Development 50 377386. doi:10.1002/(SICI)1098-2795(199808)50:4<377::AID-MRD1>3.0.CO;2-F.

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Kursawe R & Paschke R 2000 Modulation of TSHR signaling by posttranslational modifications. Trends in Endocrinology and Metabolism 18 199206. doi:10.1016/j.tem.2007.05.002.

    • Search Google Scholar
    • Export Citation
  • Miura M, Tanaka K, Komatsu Y, Suda M, Yasoda A, Sakuma Y, Ozasa A & Nakao K 2002 Thyroid hormones promote chondrocyte differentiation in mouse ATDC5 cells and stimulate endochondral ossification in fetal mouse tibias through iodothyronine deiodinases in the growth plate. Journal of Bone and Mineral Research 17 443454. doi:10.1359/jbmr.2002.17.3.443.

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S & Marshak DR 1999 Multilineage potential of adult human mesenchymal stem cells. Science 284 143147. doi:10.1126/science.284.5411.143.

    • Search Google Scholar
    • Export Citation
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    FACS analysis of specific markers for hBMSCs. Cells were stained with anti-CD13, anti-CD14, anti-CD29, anti-CD34, anti-CD73, and anti-CD105 antibodies. PE- or FITC-labeled isotype-matched antibodies were used as control. Cells from early (the 5th passage), middle (the 12th passage), and late passages (the 20th passage) were tested. Numbers at the corners represent percent values of positive cells for indicated markers.

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    Determination of TSHR expression. TSHR expression on the cell surface of hMSCs was determined by three different methods. (A) Western blot: lane 1, TSHR in human thyroid cells; lane 2, TSHR in hBMSCs from the first donor; and lane 3, TSHR in hBMSCs from the second donor. (B) In fluorescence confocal microscopy studies, cells were stained with the primary antihuman TSHR antibody and FITC-conjugated secondary Ab plus Hoechst to stain the nuclei. Inverted Leica DM4000 with 4-laser Leica confocal microscope system (Leica GE) and software, Wetzlar, Germany. (C) TSHR expression was also assessed by flow cytometry.

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    Determination of cAMP in TSH-treated hBMSCs. An ELISA-based cAMP assay was used to measure cAMP increase followed by TSH treatment. Forskolin served as a positive control. Assay was performed in triple wells per sample. The mean value and the s.d.s were calculated.

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    Regulation of gene expression of hBMSCs. (A) Metabolic markers: ATP-binding cassette sub-family G (white) member 2 (ABCG2), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), aldehyde dehydrogenase 2 family (mitochondrial) (ALDH2), fibroblast growth factor receptor 1 (FGFR1). (B) Self-renewal markers of hBMSCs: heat-shock 70 kDa protein 9 (HSPA9), MYST histone acetyltransferase 1 (MYST1), MYST histone acetyltransferase 2 (MYST2), neurogenin 2 (NEUROG2), sex-determining region Y (SRY)-box 1 (SOX1), sex-determining region Y (SRY)-box 2 (SOX2). (C) Gene expression for signaling pathways related to stem cell maintenance such as WNT pathway: adenosine deaminase RNA-specific (ADAR), adenomatous polyposis coli (APC), axin 1 (AXIN1), beta-transducin repeat containing (BTRC), cyclin D1 (CCND1), frequently rearranged in advanced T-cell lymphomas (FRAT1), frizzled homolog 1 (Drosophila) (FZD1), V-myc myelocytomatosis viral oncogene homolog (avian) (MYC), peroxisome proliferator-activated receptor delta (PPARD), wingless-type MMTV integration site family, member 1 (WNT1). (D) Notch pathway: delta-like 1 (Drosophila) (DLL1), delta-like 3 (Drosophila) (DLL3), Deltex homolog 1 (Drosophila) (DTX1), Deltex homolog 2 (Drosophila) (DTX2), Dishevelled, dsh homolog 1 (Drosophila) (DVL1), E1A binding protein p300 (EP300), GCN5 general control of amino-acid synthesis 5-like 2 (yeast) (GCN5L2 or KAT2A), histone deacetylase 2 (HDAC2), Jagged 1 (JAG1), Notch homolog 1, translocation-associated (NOTCH1), Notch homolog 2 (NOTCH2), Numb homolog (NUMB) *(P<0.05).

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    Chondrogenic-like cell differentiation and protein expression of COL2A1 and COL9A1. (A) Immunohistochemistry for COL9A1 detection. (B) Immunohistochemistry for COL2A1 detection. (C) Alcian blue staining of differentiated cells by TSH stimulation.

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    A model for associated functions with TSHR expression in hBMSCs. The TSHR is a G protein-coupled receptor. When TSH binds to its receptor, subunits of the G protein activate some biochemical pathways as shown in the figure. As a consequence of these activations, expressions of some transcription factors or some growth factors are regulated in processes of self-renewal, differentiation, and maintenance. TSH receptor (TSHR), G protein s (Gs), G protein q (Gq), protein kinase A (PKA), phosphatidyl inositol 4,5-bisphosphate (PIP2), inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG), protein kinase C (PKC), and Janus kinase 2 (JAK2).

  • Abdallah BM & Kassem M 2008 Human mesenchymal stem cells: from basic biology to clinical applications. Gene Therapy 15 109116. doi:10.1038/sj.gt.3303067.

    • Search Google Scholar
    • Export Citation
  • Agretti P, Chiovato L, De Marco G, Marcocci C, Mazzi B, Sellari-Franceschini S, Vitti P, Pinchera A & Tonacchera M 2002 Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues. European Journal of Endocrinology 147 733739. doi:10.1530/eje.0.1470733.

    • Search Google Scholar
    • Export Citation
  • Agretti P, De Marco G, De Servi M, Marcocci C, Vitti P, Pinchera A & Tonacchera M 2005 Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation. European Journal of Endocrinology 152 777784. doi:10.1530/eje.1.01900.

    • Search Google Scholar
    • Export Citation
  • Bagriacik EU & Klein JR 2000 The thyrotropin (thyroid-stimulating hormone) receptor is expressed on murine dendritic cells and on a subset of CD45RB high lymph node T cells: functional role for thyroid-stimulating hormone during immune activation. Journal of Immunology 164 61586165.

    • Search Google Scholar
    • Export Citation
  • Bell DM, Leung KK, Wheatley SC, Ng LJ, Zhou S, Ling KW, Sham MH, Koopman P, Tam PP & Cheah KS 1997 Sox9 directly regulates the type-II collagen gene. Nature Genetics 16 174178. doi:10.1038/ng0697-174.

    • Search Google Scholar
    • Export Citation
  • Bell A, Gagnon A, Grunder L, Parikh SJ, Smith TJ & Sorisky A 2000 Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts. American Journal of Physiology. Cell Physiology 279 C335C340.

    • Search Google Scholar
    • Export Citation
  • Cowman MK, Slahetka MF, Hittner DM, Kim J, Forino M & Gadelrab G 1984 Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides. Biochemical Journal 221 707716.

    • Search Google Scholar
    • Export Citation
  • Davies T, Marians R & Latif R 2002 The TSH receptor reveals itself. Journal of Clinical Investigation 110 161164.

  • Dutton CM, Joba W, Spitzweg C, Heufelder AE & Bahn RS 1997 Thyrotropin receptor expression in adrenal, kidney, and thymus. Thyroid 7 879884. doi:10.1089/thy.1997.7.879.

    • Search Google Scholar
    • Export Citation
  • Ellerhorst JA, Sendi-Naderi A, Johnson MK, Cooke CP, Dang SM & Diwan AH 2006 Human melanoma cells express functional receptors for thyroid-stimulating hormone. Endocrine-Related Cancer 13 12691277. doi:10.1677/erc.1.01239.

    • Search Google Scholar
    • Export Citation
  • Eyre DR, Pietka T, Weis MA & Wu JJ 2004 Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage. Journal of Biological Chemistry 279 25682573. doi:10.1074/jbc.M311653200.

    • Search Google Scholar
    • Export Citation
  • François S, Bensidhoum M, Mouiseddine M, Mazurier C, Allenet B, Semont A, Frick J, Saché A, Bouchet S & Thierry D et al. 2006 Local irradiation not only induces homing of human mesenchymal stem cells at exposed sites but promotes their widespread engraftment to multiple organs: a study of their quantitative distribution after irradiation damage. Stem Cells 24 10201029. doi:10.1634/stemcells.2005-0260.

    • Search Google Scholar
    • Export Citation
  • Gao F, Kwon SW, Zhao Y & Jin Y 2009 PARP1 poly (ADP-ribosylates Sox2 to control Sox2 protein levels and FGF4 expression during embryonic stem cell differentiation. Journal of Biological Chemistry 284 2226322273. doi:10.1074/jbc.M109.033118.

    • Search Google Scholar
    • Export Citation
  • Go MJ, Takenaka C & Ohgushi H 2008 Forced expression of Sox2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities. Experimental Cell Research 314 11471154. doi:10.1016/j.yexcr.2007.11.021.

    • Search Google Scholar
    • Export Citation
  • Haraguchi K, Shimura H, Lin L, Saito T, Endo T & Onaya T 1996 Functional expression of thyrotropin receptor in differentiated 3T3-L1 cells: a possible model cell line of extrathyroidal expression of thyrotropin receptor. Biochemical and Biophysical Research Communications 223 193198. doi:10.1006/bbrc.1996.0868.

    • Search Google Scholar
    • Export Citation
  • Hoermann R 1996 Stimulation of thyroidal and extrathyroidal thyrotropin receptors. Experimental and Clinical Endocrinology and Diabetes 104 (Suppl 4) 8891. doi:10.1055/s-0029-1211710.

    • Search Google Scholar
    • Export Citation
  • Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun Y & Lemischka IR 2006 Dissecting self-renewal in stem cells with RNA interference. Nature 442 533538. doi:10.1038/nature04915.

    • Search Google Scholar
    • Export Citation
  • Johnson LR, Lamb KA, Gao Q, Nowling TK & Rizzino A 1998 Role of the transcription factor Sox-2 in the expression of the FGF-4 gene in embryonal carcinoma cells. Molecular Reproduction and Development 50 377386. doi:10.1002/(SICI)1098-2795(199808)50:4<377::AID-MRD1>3.0.CO;2-F.

    • Search Google Scholar
    • Export Citation
  • Kamachi Y, Uchikawa M & Kondoh H 2000 Pairing sox off: with partners in the regulation of embryonic development. Trends in Genetics 16 182187. doi:10.1016/S0168-9525(99)01955-1.

    • Search Google Scholar
    • Export Citation
  • Katoh M & Katoh M 2007 WNT signaling pathway and stem cell signaling network. Clinical Cancer Research 13 40424045. doi:10.1158/1078-0432.CCR-06-2316.

    • Search Google Scholar
    • Export Citation
  • Kemp KC, Hows J & Donaldson C 2005 Bone marrow-derived mesenchymal stem cells. Leukemia and Lymphoma 46 15311544. doi:10.1080/10428190500215076.

  • Kim WB, Lewis CJ, McCall KD, Malgor R, Kohn AD, Moon RT & Kohn LD 2007 Overexpression of Wnt-1 in thyrocytes enhances cellular growth but suppresses transcription of the thyroperoxidase gene via different signaling mechanisms. Endocrinology 193 93106. doi:10.1677/JOE-06-0025.

    • Search Google Scholar
    • Export Citation
  • Kolf CM, Cho E & Tuan RS 2007 Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Research and Therapy 9 110. doi:10.1186/ar2116.

    • Search Google Scholar
    • Export Citation
  • Kursawe R & Paschke R 2000 Modulation of TSHR signaling by posttranslational modifications. Trends in Endocrinology and Metabolism 18 199206. doi:10.1016/j.tem.2007.05.002.

    • Search Google Scholar
    • Export Citation
  • Miura M, Tanaka K, Komatsu Y, Suda M, Yasoda A, Sakuma Y, Ozasa A & Nakao K 2002 Thyroid hormones promote chondrocyte differentiation in mouse ATDC5 cells and stimulate endochondral ossification in fetal mouse tibias through iodothyronine deiodinases in the growth plate. Journal of Bone and Mineral Research 17 443454. doi:10.1359/jbmr.2002.17.3.443.

    • Search Google Scholar
    • Export Citation
  • Niwa H, Miyazaki J & Smith AG 2000 Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genetics 24 372376. doi:10.1038/74199.

    • Search Google Scholar
    • Export Citation
  • Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S & Marshak DR 1999 Multilineage potential of adult human mesenchymal stem cells. Science 284 143147. doi:10.1126/science.284.5411.143.

    • Search Google Scholar
    • Export Citation
  • Quintarelli G, Scott JE & Dellovo MC 1964 The chemical and histochemical properties of Alcian Blue. II. Dye binding of tissue polyanions. Histochemie 4 8698. doi:10.1007/BF00306150.

    • Search Google Scholar
    • Export Citation
  • Scott JE, Quintarelli G & Dellovo MC 1964 The chemical and histochemical properties of alcian blue I. the mechanism of alcian blue staining. Histochemie 4 7385. doi:10.1007/BF00306149.

    • Search Google Scholar
    • Export Citation
  • Sellitti DF, Akamizu T, Doi SQ, Kim GH, Kariyil JT, Kopchik JJ & Koshiyama H 2000 Renal expression of two ‘thyroid-specific’ genes: thyrotropin receptor and thyroglobulin. Experimental Nephrology 8 235243. doi:10.1159/000020674.

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