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First published online February 9, 2006
Stem Cells Vol. 24 No. 6 June 2006, pp. 1487 -1495
doi:10.1634/stemcells.2005-0415; www.StemCells.com
© 2006 AlphaMed Press

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STEM CELL GENETICS AND GENOMICS

Glucocorticoids Promote Chondrogenic Differentiation of Adult Human Mesenchymal Stem Cells by Enhancing Expression of Cartilage Extracellular Matrix Genes

Assia Derfoula, Geraldine L. Perkinsa,b, David J. Halla,b, Rocky S. Tuana

a Cartilage Biology and Orthopaedics Branch,
b Cartilage Genetics Group, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, NIH, Department of Health and Human Services, Bethesda, Maryland, USA

Key Words. Differentiation • Human mesenchymal stem cells • Glucocorticoid • Cartilage

Correspondence: Rocky S. Tuan, Ph.D., Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, NIH, 50 Center Drive, Room 1523, MSC 8022, Bethesda, Maryland 20892-8022, USA. Telephone: 301-451-6854; Fax: 301-435-8017; e-mail: tuanr{at}mail.nih.gov

Received August 26, 2005; accepted for publication February 2, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the adult human, mesenchymal stem cells (hMSCs) resident in the bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. Glucocorticoids (GCs) are required for chondrogenic differentiation of hMSCs in vitro; however, the exact role of GCs in this process is not known. In this study, we examined the effects of dexamethasone (DEX) on chondrogenic differentiation of hMSCs in the presence or absence of DEX, transforming growth factor-ß (TGF-ß), or DEX plus TGF-ß. GC treatment upregulated gene expression of cartilage matrix components aggrecan, dermatopontin, and collagen type XI; enhanced TGF-ß-mediated upregulation of collagen type II and cartilage oligomeric matrix protein; and increased aggrecan and collagen type II production as well as cartilage matrix-sulfated proteoglycans as assessed by immunohistochemistry and alcian blue staining. Inclusion of an antagonist of GCs inhibited expression of chondrogenic differentiation markers, suggesting that the GC effects during chondrogenesis are mediated by the GC receptor (GR). Steady levels of the major active form of GR, GR{alpha}, were detected in both undifferentiated and differentiating hMSCs, whereas the dominant-negative isoform GRß, present at low levels in undifferentiated hMSCs, was downregulated during chondrogenesis. In the presence of DEX and TGF-ß, expression of a collagen type II gene promoter luciferase reporter construct in hMSCs was upregulated. However, coexpression of GRß dramatically inhibited promoter activity, suggesting that GR{alpha} is required for GC-mediated modulation of chondrogenesis and that GCs may play an important role in the maintenance of cartilage homeostasis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Development of long bones results from endochondral ossification, a process consisting of a series of highly regulated events that involves bone formation with a cartilage intermediate. First, mesenchymal cells proliferate and undergo condensation to form cell aggregates, followed by the differentiation of the condensed mesenchymal cells into chondrocytes, generating a highly organized cartilage-specific extracellular matrix. Chondrocytes mature into hyperthophic chondrocytes and undergo apoptosis, followed by the invasion of the cartilaginous matrix by osteoblasts, leading ultimately to bone formation [1–3]. This process is maintained during postnatal life in the growth plate of long bones, resulting in longitudinal growth, and may be reactivated during normal bone turnover and repair. This possibility is supported by the demonstrations of the persistence of mesenchymal progenitor cells in adult bone marrow, cartilage, and bone tissues, which are able to differentiate along the chondrogenic and osteogenic lineages in vitro [4–7].

Transforming growth factor-ß (TGF-ß) plays a central role during chondrogenesis [8, 9]. TGF-ß upregulates a number of molecules associated with prechondrogenic mesenchyme condensation, a critical step in the chondrogenesis process [10]. Furthermore, TGF-ß determines the commitment of hMSCs derived from bone marrow to mesenchymal chondroprogenitor cells, in vitro [4, 5]. In addition, this process requires the glucocorticoid (GC) analog dexamethasone (DEX). However, the precise mechanisms of GC action on stem cell chondrogenesis and skeletal function are not known.

Prolonged administration of pharmacological doses of synthetic glucocorticoid hormones, for the treatment of clinical disorders, results in growth suppression in children and is associated with low bone mineral density and an increase of fracture risk in adults. Possible mechanisms include interference with the growth hormone and insulin growth factor axis in children and reduced production of sex steroids and inhibition of gastrointestinal and renal Ca2+ handling in adults, but most likely impairment of proliferation and differentiation of cells of the osteoblastic lineage [11, 12]. However, anabolic effects of GCs on cells of the osteoblast lineage have been known for a long time in various cell systems in vitro. GCs have been shown to promote differentiation of osteoblasts [13–15] and chondrocytes [16, 17] and to be required for chondrogenic and osteogenic differentiation of adult human mesenchymal stem cells (hMSCs). These observations suggest that physiological GCs may be involved in the proliferation and differentiation of chondrocytes in vivo and that GCs may influence skeletal function in vivo by acting on adult mesenchymal progenitor cells.

GC function is mediated by the GC receptor (GR); GR expression has been demonstrated in both developing and adult tissues in both rodents and humans. During development, GR is detected as early as day 9.5 of mouse development, and in third-trimester human fetal tissues [18, 19]. In the rat growth plate, GR is detected in the proliferative, mature, and hypertrophic chondrocytes [20]. In humans, GR is widely distributed at sites of endochondral bone formation in resting, proliferating, mature, and hypertrophic chondrocytes of the neonatal rib and vertebrae, and it is also detected in the adult hypertrophic chondrocytes of the tibia. In bone, GR is highly expressed in osteoblasts at sites of bone remodeling but is not detected in osteoclasts [21–23]. These observations suggest that GCs may be involved in the proliferation and differentiation of chondrocytes and osteoblasts in vivo. The presence of the functional GR in human cartilage and bone in situ suggests that the actions of GCs on bone and cartilage may be mediated, in part, directly via the GR at different stages of life. This hypothesis is supported by the recent observation of reduced bone formation in transgenic mice expressing the GC-inactivating enzyme, 11-ß hydroxysteroid dehydrogenase 2 (11-ß HSD-2), transforming cortisol into cortisone, a GC with low affinity for the GR [24]. In addition, lower levels of GR were detected in chondrocytes of osteoarthritis patients compared with normal human chondrocytes, suggesting a role for physiological GCs in cartilage homeostasis [23, 25].

To test this hypothesis, we have examined the effects of the GC DEX on the chondrogenic differentiation of a human adult multipotential mesenchymal progenitor cell line (hMSC), previously established from trabecular bone [6, 7]. Here, we report that, under chondrogenic conditions, GC treatment of hMSC induced gene expression of extracellular matrix components aggrecan and collagen type XI, and enhanced TGF-ß-mediated upregulation of collagen type II and cartilage oligomeric matrix protein (COMP). Inclusion of RU 36846 (RU; Mifepristone), an antagonist of GCs, inhibited expression of cartilage markers, suggesting that GC effects during chondrogenesis are mediated by GR. GR{alpha} isoform was detected in both undifferentiated and differentiating mesenchymal stem cells (MSCs). However, expression of GRß, the dominant-negative isoform detected in undifferentiated hMSCs, was downregulated upon induction of chondrogenesis. Overexpression of the GRß dramatically inhibited a collagen type II gene promoter reporter activity, indicating that GR{alpha} activity is required for chondrogenic differentiation of mesenchymal stem cell.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials
DEX and RU were purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com). DEX was dissolved in water, and RU was reconstituted in 95% ethanol as 10–3 M stock solutions. Recombinant human TGF-ß3 from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com) was dissolved in 4 mM HCl containing 0.1% bovine serum albumin (BSA) as a 2 µg/ml stock solution.

Cell Culture
The multipotential mesenchymal progenitor cell line used in this study was previously established in our laboratory and was derived from human adult trabecular bone and immortalized by transduction with human papillomavirus oncoproteins E6/E7 [26]. hMSCs were grown in monolayer culture in high-glucose Dulbecco’s modified Eagle’s medium (BioWhittaker, Walkersville, MD, http://www.bmaproducts.com) containing L-glutamine and 1.5 g/l sodium bicarbonate and supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA, http://www.atlantabio.com) and antiobiotic-antimycotic solutions (10,000 U of penicillin, 25 µg of amphotericin B, 10,000 µg of streptomycin) from Gibco (Grand Island, NY, http://www.invitrogen.com). For chondrogenic cultures, hMSCs were grown as high-density cell pellets by spinning down 2.5 x 105 cells in chondrogenic medium consisting of serum-free medium containing insulin-transferrin-selenious acid mix (ITS) from BD Biosciences (Bedford, MA, http://www.bdbiosciences.com), 100 mg/ml sodium pyruvate, 40 µg/ml L-proline, and 50 µg/ml L-ascorbic acid 2-phosphate. Cultures were carried out for 1, 11, or 21 days in the presence or absence of 100 nM DEX or TGF-ß3 at a final concentration of 10 ng/ml, as well as a combination of both DEX and TGF-ß3. Control cultures consisted of cells incubated in chondrogenic medium without DEX or TGF-ß3. Pellet cultures were processed for RNA isolation followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis performed according to standard procedures or fixed in 4% paraformaldehyde for histochemical and immunohistochemical analyses. To control for the effect of cell aggregation in cell pellets, hMSCs were grown as monolayer cultures at 7.5 x 103 cells per cm2 in FBS-containing medium or in serum-free chondrogenic medium without DEX or TGF-ß3 and were processed for RNA isolation followed by RT-PCR analyses performed according to standard procedures.

Plasmids, Transfections, and Reporter Assay Analyses
The collagen type II reporter construct (pColl II-Luc), obtained from Dr. Mary Goldring [27], consists of 4 kilobases of the 5'-flanking sequence of the human procollagen type II {alpha}1 gene (COL2A1, –577/+3428), which encompasses the promoter region, exon 1, and a putative enhancer sequence in the first intron, inserted upstream of the luciferase reporter gene in the pGL-3 basic vector (Promega, Madison, WI, http://www.promega.com). The pF25-GRß was obtained from Dr. George P. Chrousos (National Institute of Child Health and Human Development, NIH), and pFlag-Sox-9 was a generous gift from Dr. Benoit de Crombrugghe. All transfection experiments were initiated on 50% confluent monolayer cultures. Cells were transfected with 2 µg of the collagen type II reporter construct in the presence or absence of 1 µg of the human GR plasmid, using the Saint mix transfection cocktail (GeneExpression Systems, Waltham, MA, http://www.expressgenes.com) according to the manufacturer’s procedure. Twelve to 18 hours post-transfection, cells were rinsed twice with phosphate-buffered saline and then treated in ITS chondrogenic medium with 100 nM DEX and 10 ng/ml TGF-ß3. Following 48 hours of treatment, cells were scraped, and cell extracts were prepared and processed for measurement of promoter activity using the luciferase assay system (Promega), according to the manufacturer’s recommendations. In these experiments, 1 µg of pcDNA-LacZ (Promega) was cotransfected to normalize for differences in transfection efficiency. ß-Galactosidase activity was measured using a luminescent ß-galactosidase detection kit, according to the manufacturer’s procedure (BD Biosciences). Statistical analyses were performed using three individual experiments performed in duplicate and the mean ± SD (p ≤ .05) was determined by analysis of variance.

RT-PCR Analyses
Total RNA was prepared from monolayer and pellet cultures using Trizol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). RNA (0.5–1 µg) was reverse-transcribed then amplified using a one-step RT-PCR procedure (Invitrogen). Reverse transcription reactions were performed at 55°C for 30 minutes and terminated by incubation at 94°C for 2 minutes. PCRs were carried out for 40 cycles of amplification, consisting of 94°C for 30 seconds, 55°C–60°C for 30 seconds, and 72°C for 45 seconds. Specific primers for the genes examined were designed based on their GenBank sequence. The following primer sets were used: 5'-TGA CCA CTT TAC TCT GGG TTT TCG-3' and 5'-ACA CGA TGC CTT TCA CCA CG-3' for aggrecan; 5'-CCG CGG TGA GCC ATG ATT CG-3' and 5'-CAG GCC CAG GAG GTC CTT TGG G-3' for collagen II; 5'-GGA AAG GAC GAA GTT GGT CTG C-3' and 5'-TTC TCC ACG CTG ATT GCT ACC C-3' for collagen XI; 5'-CAA CTG TCCCCA GAA GAG CAA-3' and 5'-TGG TAG CCA AAG ATG AAG CCC-3' for COMP; 5'-TGG ACC TCA GTC TTC TCT GGG-3' and 5'-TCC TAG CTA GCT TCA GAG CCG-3' for dermatopontin; 5'-AGA GGA GGA GCT ACT GTG AAG-3' and 5'-GGT CGA CCT ATT GAG GTT TGC-3' for GR{alpha}; 5'-CCT AAG GAC GGT CTG AAG AGC-3' and 5'-CCA CGT ATC CTA AAA GGG CAC-3' for GRß; 5'-CTC GAG TCG GAT GGC TTT TTA TG-3' and 5'-ACT TGC TTG CAG AAT AGG-3' for 11-ß HSD-1; and 5'-ACC ACA GTC CAT GCC ATC AC-3' and 5'-TCC ACC ACC CTG TTG CTG TA-3' for glyceraldehyde-3-phospho-dehydrogenase.

Histological and Immunohistochemical Analyses
Pellet cultures were fixed in 4% paraformaldehyde, dehydrated using a graded series of ethanol washes, and embedded in paraffin. Sections 8 µm in thickness were stained with eosin or alcian blue. For immunohistochemical analyses, sections were digested with hyaluronidase (Sigma-Aldrich) or 20 µg/ml proteinase K (Roche Diagnostics, Indianapolis, http://www.roche-applied-science.com) in 10 mM Tris-HCl (PH 7.5) for 30 min at 37°C and then incubated overnight at room temperature with the indicated antibodies in Tris-buffered saline containing 0.1% BSA. Specific antibodies to aggrecan and collagen type II were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA, http://www.uiowa.edu/~dshbwww). Antibodies to human GR (GR{alpha} and GRß) were purchased from Affinity Bioreagents (Golden, CO). Immunostaining was detected histochemically using the streptavidin-peroxidase Histostain SP Kit for DAB (Zymed Laboratories, San Francisco), and in the indicated experiments, cells were counterstained with hematoxylin. Cells were examined under a Leica microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com) at the indicated magnification.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
GCs Positively Regulate Chondrogenic Marker Gene Expression
Upon DEX and TGF-ß treatment of high-density pellet cultures of hMSCs, pellets increased more than 3 times in diameter compared with untreated pellets (Fig. 1AGo). Treated pellets showed significant accumulation of sulfated proteoglycans in the matrix, as demonstrated by histological staining with alcian blue of sections derived from 3-week-old pellet cultures (Fig. 1AGo). RT-PCR analyses showed that treatment of high-density pellet cultures with DEX and TGF-ß induced mRNA expression of aggrecan, COMP, and cartilage-associated collagens, such as collagen types II and XI (Col II, Col XI), compared with untreated control pellet hMSCs (Fig. 1AGo). These cartilage marker genes were also not detected in monolayer hMSCs grown either in FBS or ITS-containing medium (Fig. 1BGo). These results indicate that the immortalized hMSCs retained the potential for chondrogenic differentiation and are responsive to induction to become chondrocytes.


Figure 1
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  		Figure 1. Chondrogenenic differentiation of human mesenchymal stem cells (hMSCs). (A): Histology. hMSCs grown for 21 days as high-density pellet cultures and treated in the presence or absence of 10 ng/ml TGF-ß and 100 nM DEX were fixed in 4% paraformaldehyde, embedded, sectioned, stained with eosin or alcian blue, and observed with bright-field microscopy at low (top) or high (bottom) magnifications. Significantly larger size of pellet and positive sulfated glycosaminoglycan staining by alcian blue were seen in pellet cultures treated with DEX and TGF-ß compared with untreated cultures. (B): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of gene expression. Primers specific for cartilage matrix genes aggrecan, Col II, Col XI, and COMP, as well as G3PDH, were used to analyze hMSCs grown in monolayer or as high-density pellet cultures and treated with DEX and TGF-ß. Electrophoretic analysis of RT-PCR products showed that expression of these cartilage matrix genes was absent or present at low levels in monolayer as well as in untreated pellet cultures but significantly upregulated in pellet cultures treated with DEX and TGF-ß. Abbreviations: Col II, collagen type II; Col XI, collagen type XI; COMP, cartilage oligomeric matrix protein; DEX, dexamethasone; FBS, fetal bovine serum; G3PDH, glyceraldehyde-3-phospho-dehydrogenase; ITS, insulin-transferrin-selenious acid mix; TGF-ß, transforming growth factor-ß.

 
We next examined the role of GCs in this differentiation process, by RT-PCR analysis of pellet cultures grown for 1 or 21 days, in the presence or absence of DEX and/or TGF-ß supplementation. Treatment of pellet cultures with DEX alone had little effect on aggrecan gene expression compared with control untreated pellets at day 1, and expression was downregulated at day 21 (Figs. 1Go, 2Go), whereas treatment with both DEX and TGF-ß induced a strong upregulation of aggrecan gene expression, beginning on day 1 and maintained up to day 21 (Fig. 2Go). Upon DEX treatment, the expression of the small proteoglycan dermatopontin and Col XI was upregulated as early as day 1 and maintained up to day 21 (Fig. 2Go). However, DEX alone had little effect on induction of COMP and Col II gene expression. In comparison, upon treatment with TGF-ß, expression of dermatopontin, Col II, Col XI, and COMP was rapidly induced (Fig. 2Go). In combination, DEX and TGF-ß treatment further enhanced DEX-mediated upregulation of dermatopontin and Col XI expression, as well as the TGF-ß-mediated upregulation of COMP, dermatopontin, and Col XI. Furthermore, the expression of aggrecan and Col II (both IIA and IIB alternatively spliced mRNA isoforms) was also upregulated (Fig. 2Go).


Figure 2
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  		Figure 2. Glucocorticoid regulation of cartilage marker gene expression in high-density pellet culture of hMSCs. (A): Reverse transcription-polymerase chain reaction (RT-PCR) analysis. Total RNA was prepared from high-density pellet cultures of human mesenchymal stem cells at culture day 1 or culture day 21, grown in either control, DEX, TGF-ß, or DEX+TGF-ß. RT-PCR analysis was performed for aggrecan, dermatopontin, Col II, Col XI, and COMP, with G3PDH as an internal control. DEX enhanced expression of Col XI and dermatopontin but had little effect on expression of aggrecan, Col II, and COMP, which were strongly upregulated by TGF-ß. DEX enhanced TGF-ß-mediated regulation of aggrecan, Col II, and COMP gene expression. Abbreviations: IIA, Col IIA transcript; IIB, Col IIB transcript; Col II, collagen type II; Col XI, collagen type XI; COMP, cartilage oligomeric matrix protein; control, serum-free medium containing insulin-transferrin-selenious acid mix only; DEX, insulin-transferrin-selenious acid mix medium supplemented with 100 nM dexamethasone; DEX+TGF-ß, insulin-transferrin-selenious acid mix medium supplemented with 100 nM dexamethasone and 10 ng/ml transforming growth factor-ß; G3PDH, glyceraldehyde-3-phospho-dehydrogenase; TGF-ß, insulin-transferrin-selenious acid mix medium supplemented with 10 ng/ml transforming growth factor-ß.

 
To confirm the effects of GCs on the expression of chondrogenic markers, we next examined aggrecan and collagen type II protein levels in sections of day 21 pellet cultures by immunohistochemistry and sulfated proteoglycan accumulation by alcian blue histochemistry. As shown in Figure 3Go, treatment of pellet cultures with DEX increased aggrecan and collagen type II levels compared with untreated controls. Similar increase of collagen type II was observed in cells treated with TGF-ß alone. These effects were also observed when cultures were treated with both DEX and TGF-ß (Fig. 3Go). Alcian blue staining further confirmed the immunohistochemical findings, with DEX and TGF-ß co-treatment producing high levels of blue stained sulfated proteoglycansin the matrix, characteristic of cartilage, whereas DEX or TGF-ß treatment alone yielded lower staining intensity compared with the untreated control, which was alcian blue-negative (Fig. 3Go). Taken together, these results show that during chondrogenic differentiation, GCs upregulate cartilage matrix genes at both the message level and the protein level. The effect of GCs is achieved through either direct regulation and/or enhancement of TGF-ß-mediated regulation of gene expression of cartilage collagen types and proteoglycans, suggesting that GCs and TGF-ß may regulate common pathways that are activated during chondrogenesis and that GCs and TGF-ß interact positively to enhance this regulation.


Figure 3
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  		Figure 3. Histochemical and immunohistochemical analyses of glucocorticoid-mediated regulation of chondrogenic marker gene expression in human mesenchymal stem cell pellet cultures. Day 21 cultures grown in either control, DEX, TGF-ß, or DEX+TGF-ß were fixed, and sections were immunostained for aggrecan and collagen type II (counterstained with hematoxylin) or directly stained with alcian blue. Aggrecan and collagen type II staining was significantly increased by DEX or TGF-ß treatment alone compared with control. Cotreatment with DEX and TGF-ß resulted in the highest increase in the levels of aggrecan and collagen type II, as well of extracellular matrix-sulfated proteoglycans, as shown by alcian blue staining. Abbreviations: control, serum-free medium containing insulin-transferrin-selenious acid mix only; DEX, insulin-transferrin-selenious acid mix medium supplemented with 100 nM dexamethasone; DEX+TGF-ß, insulin-transferrin-selenious acid mix medium supplemented with 100 nM dexamethasone and 10 ng/ml transforming growth factor-ß; TGF-ß, insulin-transferrin-selenious acid mix medium supplemented with 10 ng/ml transforming growth factor-ß.

 
GC Regulation of Chondrogenesis Is Mediated by GR
To assess whether GC effects on chondrogenic marker expression were mediated by GR, we tested the effect of RU, the GC antagonist, on DEX-mediated gene expression. RU is known to bind GR with similar affinity to DEX, but it produces a GR-RU complex that is unable to activate expression of GR target genes. Pellet cultures treated with DEX and/or a 10-fold excess of RU were carried out for 11 or 21 days. As shown in Figure 4AGo, RT-PCR analysis of total RNA prepared from day-11 pellets revealed that DEX enhanced expression of aggrecan, dermatopontin, and Col XI, but DEX-induced upregulation was inhibited by RU cotreatment; RU treatment alone resulted in gene expression levels somewhat similar to those detected in control untreated cultures (Fig. 4AGo). Similar inhibition by RU of DEX-induced chondrogenic marker gene expression was observed at day 21 for Col XI and dermatopontin (data not shown). The effects of RU were confirmed by immunohistochemistry of 21-day pellet cultures (Fig. 4BGo), showing that RU cotreatment inhibited DEX-mediated increased level of collagen type XI. Treatment with RU alone had little effect on collagen type II levels, the immunostaining intensity being slightly lower than basal levels detected in untreated control. Taken together, these results indicate that GC effects on mesenchymal chondrogenesis are specifically mediated by the GR.


Figure 4
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  		Figure 4. Glucocorticoid (GC) regulation of cartilage extracellular marker gene expression is mediated by the GC receptor. (A): Reverse transcription-polymerase chain reaction (RT-PCR). Day 11 high-density pellet cultures of human mesenchymal stem cells (hMSCs) grown in either control, DEX, RU, or DEX+RU were analyzed by RT-PCR. DEX mediated upregulation of aggrecan dermatopontin, and Col XI was inhibited by co-treatment with RU, indicating that GC effects during chondrogenesis are GC receptor-mediated. (B): Immunohistochemistry. hMSCs were grown as described in (A) for 21 days, and sections were immunostained for collagen type II and counterstained with hematoxylin. DEX treatment significantly enhanced collagen type II production, an effect that was inhibited by cotreatment with RU, whereas RU alone had no detectable effect. Abbreviations: Col XI, Col XI, collagen type XI; control, serum-free medium containing insulin-transferrin-selenious acid mix only; DEX, insulin-transferrin-selenious acid mix medium supplemented with 100 nM dexamethasone; DEX+RU, insulin-transferrin-selenious acid mix medium supplemented with 100 nM dexamethasone and 1 µM GC antagonist RU 36846; G3PDH, glyceraldehyde-3-phospho-dehydrogenase; RU, insulin-transferrin-selenious acid mix medium supplemented with 1 µM GC antagonist RU 36846.

 
To further demonstrate the involvement of receptor-mediated GC signaling during chondrogenic differentiation of hMSCs, we examined the expression of GR{alpha} in sections of pellet cultures by immunohistochemistry. As shown in Figure 5Go, GR{alpha} was detected in the cytoplasm and nucleus of untreated cultures and appeared to accumulate in the nucleus upon treatment with DEX alone and DEX and TGF-ß combined, suggesting that the GC signaling pathway is active during chondrogenesis.


Figure 5
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  		Figure 5. Detection of glucocorticoid receptor (GR) in human mesenchymal stem cell (hMSCs) and its nuclear translocation upon DEX treatment during chondrogenic differentiation. hMSCs were grown for 21 days as high-density pellet cultures in either control, DEX, or DEX+TGF-ß. Histological sections were immunostained for GR{alpha}. In control, untreated pellets, abundant GR{alpha} was detected, present in both the cytoplasm and nucleus. Upon DEX treatment, GR{alpha} accumulated in the nucleus, consistent with its nuclear translocation in both DEX-treated and DEX+TGF-ß-treated pellets. Omission of primary antibodies resulted in no staining. Abbreviations: Ab, antibody; control, serum-free medium containing insulin-transferrin-selenious acid mix only; DEX, insulin-transferrin-selenious acid mix medium supplemented with 100 nM dexamethasone; DEX+TGF-ß, DEX, insulin-transferrin-selenious acid mix medium supplemented with 100 nM dexamethasone and transforming growth factor-ß; TGF-ß, 10 ng/ml transforming growth factor-ß.

 
Modulation of GR Levels During Chondrogenesis
To analyze the involvement of GR in hMSC chondrogenesis, we next examined GR{alpha} and GRß gene expression in undifferentiated hMSCs as well as during their chondrogenesis. GR{alpha} constitutes the major active GR form, whereas GRß, an alternatively spliced form, constitutes a dominant-negative isoform of GR{alpha} [28, 29]. Both GR{alpha} and GRß were detected in undifferentiated hMSCs in monolayer cultures (Fig. 6AGo). In pellet cultures of hMSCs undergoing chondrogenic differentiation, steady levels of GR{alpha} were detected, independent of DEX and TGF-ß treatment (Fig. 6BGo). In addition, 11-ß HSD-1, a GC-metabolizing enzyme that converts the inactive GC cortisone to the active form cortisol, was expressed at low levels in undifferentiated hMSCs monolayer cultures. Expression of 11-ß HSD-1 was enhanced by DEX and TGF-ß co-treatment during early chondrogenesis, compared with DEX treatment alone at day 1 and day 11 (Fig. 6BGo). Enhanced levels of 11-ß HSD-1 suggest availability of higher levels of active GCs during chondrogenesis. Interestingly, in pellet cultures of hMSCs upon DEX and TGF-ß co-treatment and induction of differentiation, expression of GRß was inhibited (Fig. 6BGo). Similar inhibition of GRß was detected in monolayer cultures treated with DEX and TGF-ß (Fig. 6AGo). Immunohistochemical detection of GRß showed lower GRß protein levels in pellet cultures treated with DEX alone or DEX and TGF-ß (Fig. 6CGo). Taken together, these observations suggest that GC effects on hMSCs chondrogenesis are mediated by steady levels of GR{alpha}. Because of the established dominant-negative effect of GRß on the GR function, down-regulation of GRß levels by DEX and TGF-ß co-treatment may be a key mechanism in GC effect on chondrogenic differentiation of hMSCs.


Figure 6
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  		Figure 6. Downregulation of GRß expression during chondrogenesis of hMSCs. (A): Monolayer cultures. Human mesenchymal stem cell (hMSC) cultures were treated for 24 hours in either ITS, DEX, or DEX+TGF-ß. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to analyze expression of GR{alpha}, GRß, and the glucocorticoid-metabolizing enzyme 11-ß hydroxysteroid dehydrogenase type 1 (11-ß HSD-1), as well as G3PDH as an internal control. The results show that GR{alpha}, GRß, and 11-ß HSD-1 are all expressed in undifferentiated hMSCs. Upon DEX treatment, GR{alpha} and GRß levels remained similar to those detected in control cells. However, GRß expression was downregulated by DEX and TGF-ß co-treatment. (B): Pellet cultures. hMSC cultures were treated with DEX or DEX plus TGF-ß for 1 or 11 days. RT-PCR analysis was performed as in (A). Steady levels of GR{alpha} were expressed during chondrogenesis. However, GRß, which was present at low levels in hMSCs, was downregulated by TGF-ß during chondrogenic differentiation in pellet cultures treated with DEX and TGF-ß, whereas 11-ß HSD-1 gene expression was enhanced by DEX and TGF-ß co-treatment. (C): Immunohistochemistry. hMSCs grown for 21 days as high-density pellet cultures in either control, DEX, TGF-ß, or DEX+TGF-ß were sectioned and immunostained for GRß. The data show specific reduction in GRß in differentiated pellets treated with both DEX and TGF-ß. Abbreviations: control, serum-free medium containing insulin-transferrin-selenious acid mix only; DEX, insulin-transferrin-selenious acid mix medium supplemented with 100 nM DEX; DEX+TGF-ß, insulin-transferrin-selenious acid mix medium supplemented with 100 nM DEX and 10 ng/ml transforming growth factor-ß; G3PDH, glyceraldehyde-3-phospho-dehydrogenase; GR, glucocorticoid receptor; ITS, serum-free medium containing insulin-transferrin-selenious acid mix only; TGF-ß, 10 ng/ml transforming growth factor-ß.

 
Inhibition of Collagen Type II Gene Promoter Reporter Activity by GRß Chondrogenic Differentiation of hMSCs
To demonstrate the specific requirement of GR function for chondrogenic differentiation, hMSCs were transfected with the human Col II gene promoter luciferase construct (pCOL2A1-luc), a chondrogenesis marker gene promoter reporter construct, in the presence or absence of the human GRß cDNA expression plasmid. At 72 hours post-transfection, the pCOL2A1-promoter activity was induced threefold in the presence of DEX and TGF-ß compared with pGL-3-Luc empty vector (Fig. 7Go). This activation was further enhanced by Sox-9 coexpression, a known positive regulator of chondrogenesis. However, activation of the Col II promoter was inhibited by more than fivefold when GRß was co-expressed, and additional Sox-9 expression was unable to block this inhibition, demonstrating the dominant-negative effect of GRß on the activity of the endogenous GR{alpha} (Fig. 7Go). The transcriptional induction of COL2A1 promoter by endogenous GR{alpha}, in the presence of DEX and TGF-ß, thus confirms that induction and/or enhancement of chondrogenesis by GCs involves transcriptional regulation of chondrogenesis marker genes. Inhibition of this regulation by GRß demonstrates the requirement of endogenous GR{alpha} activity for the enhancement of TGF-ß-mediated chondrogenesis.


Figure 7
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  		Figure 7. Inhibition of collagen II (Col II) promoter reporter activity by GRß overexpression. human mesenchymal stem cells (hMSCs) grown as monolayer cultures in serum-containing medium were transfected with the human Col II promoter reporter construct or with the pGL-3 basic control reporter construct driving luciferase expression. In the indicated experiments, the human GRß expression plasmid was co-transfected. Other cultures were transfected with Sox-9 expression plasmid. Following 18 hours of transfection, cells were washed with phosphate-buffered saline and transferred to serum-free medium containing insulin-transferrin-selenious acid mix supplemented with 100 nM dexamethasone and 10 ng/ml transforming growth factor-ß, and luciferase activity was measured in cell extracts at 48 hours post-treatment. The data showed a fivefold inhibition of the Col II promoter in the presence of GRß. As expected, co-transfection of Sox-9 expression plasmid resulted in very strong activation of the Col II promoter. Expression of GRß inhibited Col II promoter activity and blocked the Sox-9 effect. The data represent the mean ± SD (p ≤ .05), as determined by analysis of variance, of three individual experiments performed in duplicate, and luciferase activity was normalized for variations in transfection efficiency on the basis of ß-galactosidase activity. Abbreviations: GR, glucocorticoid receptor; pCol-II, Col II promoter reporter construct; pGL-3, pGL-3 basic control reporter construct.

 

   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
GCs are used for the differentiation of hMSCs into multiple lineages in vitro, including stroma, fat, muscle, bone, and cartilage. However, the mechanism of GC action during these differentiation processes is not well understood. In this study, we have shown that GCs play an important role in the chondrogenic differentiation of hMSCs in vitro. Our data show that GCs promote chondrogenesis by directly regulating the expression of cartilage extracellular matrix genes and/or enhancing TGF-ß-mediated upregulation of their expression.

GCs are known to promote cell differentiation in various systems both in vivo and in vitro. During development, GCs play an important role in lung, liver, and adrenal morphogenesis. GR–/– mice die shortly after birth from respiratory failure due to impaired lung maturation. In addition, these animals display lack of gluconeogenesis in the liver and impaired differentiation and function of the adrenal medulla, as well as impaired proliferation of the erythroid precursors of the bone marrow [30]. GC effects on cartilage and bone in vivo are not well-known and have not been evaluated in GR–/– mice. However, disruption of GC signaling in cells of the osteoblast lineage in mice has recently provided a means to determine the role of endogenous GCs in bone [31, 32]. Mice deficient in 11-ß HSD-1, a reductase transforming the inactive form of GCs, cortisone, to cortisol, lack bone marrow adipocytes but maintain normal bone formation [33]. However, transgenic mice expressing the GC-inactivating enzyme, 11-ß HSD-2, under the control of collagen type 1 gene promoter displayed reduced bone formation [24], suggesting a positive role of GCs on osteoblast differentiation. In vitro, GCs have also been shown to stimulate proteoglycan synthesis in chondrocytes [16]. Positive GC effects on differentiation of cells of the osteoblastic lineage have been reported in rodent calvarial cells and organoid cultures [14, 15, 17, 34], as well as bone marrow MSCs [4, 5, 35, 36]. Our findings extend these observations to the chondrogenic differentiation of hMSCs, an important step in the osteochondral differentiation process. Because this process remains active during growth, bone homeostasis and repair, these findings support an anabolic effect of GCs on skeletal function.

In our study, DEX upregulated gene expression and protein levels of several cartilage matrix markers, in particular Col XI. However, DEX alone had little effect on aggrecan, COMP, and Col II, whose expression was dependent on TGF-ß, but DEX enhanced their TGF-ß-mediated expression. Interestingly, DEX treatment enhanced aggrecan and Col II protein levels, suggesting that DEX enhances cartilage phenotype through an indirect mechanism. Taken together, these results suggest positive interactions between TGF-ß and GC signaling pathways in the modulation of their function during chondrogenesis. That GC and TGF-ß interact both positively and negatively and that GR directly interacts with the TGF-ß signaling molecule, Smad3, have been reported [37, 38]. Moreover, our results showed that the GC effects on the chondrogenic differentiation of hMSCs are mediated in a receptor-dependent manner, since a specific GC antagonist RU36846, blocked DEX from positively regulating chondrogenic marker expression.

Our study is, to our knowledge, the first to show expression of GR in hMSCs and to establish the presence of a functional GC signaling pathway in these cells. This was demonstrated by the detection of both cytoplasmic and nuclear localization of GR in the absence of GCs and its accumulation into the nucleus in a hormone dependent-manner, as established for other GC target cell systems [39, 40]. Our data show that GR mRNA and protein are expressed in both undifferentiated hMSCs and in hMSCs undergoing chondrogenesis. The level of the major active GR isoform, GR{alpha}, was unchanged in both undifferentiated hMSCs and in cells undergoing chondrogenesis. GR{alpha} is known to be the major GR isoform mediating GC effects in various target tissues. In addition, low levels of GRß, the alternatively spliced form, were detected in undifferentiated hMSCs, with both cytoplasmic and nuclear localization that was not sensitive to DEX treatment (data not shown). GRß is unable to bind GC hormones due to the lack of 15 amino acids critical for ligand binding, and it constitutes a dominant-negative form of GR [28, 29, 41]. GRß levels were downregulated in hMSCs during early stages of chondrogenic differentiation upon DEX and TGF-ß co-treatment, but not by DEX treatment alone, suggesting that GRß is negatively regulated by TGF-ß. In addition, overexpression of GRß in hMSCs efficiently blocked the activation of Col II gene promoter activation by DEX+TGF-ß. GRß has been shown to inhibit GR-mediated regulation of gene expression in vitro, and high levels of GRß have been associated with GC resistance in vivo [29, 42], suggesting that GRß may constitute a naturally occurring dominant-negative inhibitor of GR{alpha} activity. Regulation of GR{alpha} function through TGF-ß-mediated changes in GRß levels during chondrogenic differentiation of hMSCs may represent a key mechanism in the control of cell differentiation and maintenance of the chondrocyte phenotype. Interestingly, decreased levels of GR{alpha} have been reported in chondrocytes of osteoarthritis patients, lending support for a role for physiological GC signaling playing a role in the maintenance of homeostasis in human adult cartilage [23, 25].

In summary, our findings demonstrate that GCs act as a key regulator of in chondrogenic differentiation of adult hMSCs. GCs promote chondrogenesis of hMSCs by inducing expression of cartilage matrix genes, mediated by GR, which is functional throughout chondrogenesis. These findings underscore the importance of physiological GCs in bone and cartilage homeostasis. Understanding the mechanism of GC action in cartilage and bone formation should shed light on how inflammatory diseases may affect skeletal function and repair.


   DISCLOSURES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
This work was supported by the NIH intramural program.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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