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 
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.
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.
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.
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.
Induction of gene expression for stem cell specific markers
| Fold change | ||||
|---|---|---|---|---|
| Genbank | Function | 10−7 Ma | 10−8 Ma | |
| Genes | ||||
| Cell adhesion molecules | ||||
| APC | Adenomatous polyposis coli | +1.02 | −1.17 | |
| BGLAP | Bone gamma-carboxyglutamate (gla) protein (osteocalcin) | +1.18 | −1.25 | |
| CD44 | CD44 molecule (Indian blood group) | +1.0 | −1.33 | |
| CDH1 | Cadherin 1, type 1, E-cadherin (epithelial) | +1.2 | +1.71 | |
| CDH2 | Cadherin 2, type 1, N-cadherin (neuronal) | +1.12 | +1.04 | |
| CTNNA1 | Catenin (cadherin-associated protein), alpha 1, 102 kDa | +1.03 | +1.01 | |
| CXCL12 | Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) | −1.36 | −1.56 | |
| NCAM1 | Neural cell adhesion molecule 1 | −1.19 | +1.05 | |
| Chromosome and chromatin modulators | ||||
| KAT2A | General control of amino-acid synthesis 5-like 2 (yeast) | −1.39 | −1.14 | |
| HDAC2 | Histone deacetylase 2 | +1.38 | +1.67 | |
| MYST1 | MYST histone acetyltransferase 1 | −1.04 | +1.02 | |
| MYST2 | MYST histone acetyltransferase 2 | −1.37 | −1.38 | |
| RB1 | Retinoblastoma 1 (including osteosarcoma) | −1.09 | −1.05 | |
| TERT | TERT Telomerase reverse transcriptase | +2.35* | +5.80* | |
| Cell cycle regulators | ||||
| AXIN1 | Axin 1 | −1.54 | −3.34* | |
| CCNA2 | Cyclin A2 | +1.11 | +1.07 | |
| CCND1 | Cyclin D1 | −1.30 | +1.43 | |
| CCND2 | Cyclin D2 | −1.08 | +1.13 | |
| CCNE1 | Cyclin E1 | −1.30 | −1.33 | |
| CDK1 | Cell division cycle 2, G1 to S and G2 to M | +1.07 | +1.10 | |
| CDC42 | Cell division cycle 42 (GTP binding protein, 25 kDa) | −1.24 | −1.27 | |
| EP300 | E1A binding protein p300 | −1.39 | −2.43* | |
| MYC | V-myc myelocytomatosis viral oncogene homolog (avian) | −1.19 | −2.95* | |
| PARD6A | Par-6 partitioning defective 6 homolog alpha (Caenorhabditis elegans) | −1.23 | −1.29 | |
| Cytokines and growth factors | ||||
| BMP1 | Bone morphogenetic protein 1 | −1.41 | −1.68 | |
| BMP2 | Bone morphogenetic protein 2 | −1.02 | +1.45 | |
| BMP3 | Bone morphogenetic protein 3 (osteogenic) | +2.90* | +6.31* | |
| CXCL12 | Chemokine (C-X-C motif) ligand 12 | −1.38 | −1.56 | |
| FGF2 | Fibroblast growth factor 2 (basic) | −1.43 | −1.44 | |
| FGF3 | Fibroblast growth factor 3 | +6.31* | +18.35* | |
| FGF4 | Fibroblast growth factor 4 | +11.37* | +31.51* | |
| GDF2 | Growth differentiation factor 2 | +3.06* | +9.76* | |
| GDF3 | Growth differentiation factor 3 | +4.78* | +18.73* | |
| IGF1 | IGF1 (somatomedin C) | −1.08 | +1.23 | |
| JAG1 | Jagged 1 (Alagille syndrome) | −1.96 | −2.14* | |
| Genes regulating cell–cell communication | ||||
| DHH | Desert hedgehog homolog (Drosophila) | +1.5 | +3.06* | |
| DLL1 | Delta-like 1 (Drosophila) | +1.5 | +2.54* | |
| GJA1 | Gap junction protein, alpha 1, 43 kDa | −1.19 | −1.18 | |
| GJB1 | Gap junction protein, beta 1, 32 kDa | +2.54* | +4.78* | |
| GJB2 | Gap 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.
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.
Induction of gene expression for stem cell differentiation markers
| Fold change | ||||
|---|---|---|---|---|
| Genbank | Function | 10−7 Ma | 10−8 Ma | |
| Genes | ||||
| Mesenchymal cell lineage markers | ||||
| ACAN | Aggrecan | −1.45 | −3.53* | |
| ALPI | Alkaline phosphatase, intestinal | −226.28* | −103.39* | |
| BGLAP | Bone gamma-carboxyglutamate (gla) protein (osteocalcin) | +1.18 | −1.25 | |
| COL1A1 | Collagen, type I, alpha 1 | −1.61 | −1.59 | |
| COL2A1 | Collagen, type II, alpha 1 | +12.71* | +25.59* | |
| COL9A1 | Collagen, type IX, alpha 1 | +4.22* | +7.45* | |
| PPARG | Peroxisome proliferator-activated receptor gamma | +1.32 | +1.96 | |
| Neural cell lineage markers | ||||
| NCAM1 | Neural cell adhesion molecule 1 | −1.19 | +1.05 | |
| OPRS1 | Opioid receptor, sigma 1 | +1.37 | −1.04 | |
| S100B | S100 calcium binding protein B | +2.90* | +3.45* | |
| TUBB3 | Tubulin, beta 3 | −1.11 | −1.45 | |
| Embryonic cell lineage markers | ||||
| ACTC1 | Actin, alpha, cardiac muscle 1 | +7.66* | +13.81* | |
| ASCL2 | Achaete–scute complex homolog 2 (Drosophila) | +4.88* | +9.11* | |
| FOXA2 | Forkhead box A2 | +1.12 | +2.21* | |
| PDX1 | Pancreatic and duodenal homeobox 1 | +4.22* | +7.40* | |
| ISL1 | ISL LIM homeobox 1 | +1.67 | +2.82* | |
| KRT15 | Keratin 15 | +2.08* | +3.47* | |
| MSX1 | Msh homeobox 1 | +1.10 | −1.04 | |
| MYOD1 | Myogenic differentiation 1 | +2.05* | +7.98* | |
| T | T, brachyury homolog (mouse) | +2.59* | +5.49* | |
*P<0.05.
TSH concentrations used to stimulate hBMSCs.
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.
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.
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