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Expression of Cyclin-Dependent Kinase Inhibitors in Epiphyseal Chondrocytes Induced to Terminally Differentiate with Thyroid Hormone

Pediatric Orthopedic Research Laboratory, Department of Orthopedics, Rainbow Babies and Childrens Hospital, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: R. Tracy Ballock, M.D., 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: rtracy.ballock{at}uhhs.com

	Abstract

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A growing body of evidence suggests that systemic hormones and peptide growth factors may exert their effects on cell growth and differentiation in part through regulation of the cell division cycle. We hypothesized that thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through controlling cell cycle progression at the G₁/S restriction point. Our results support this hypothesis by demonstrating that treatment of epiphyseal chondrocytes with thyroid hormone under chemically defined conditions results in the arrest of DNA synthesis and the onset of terminal differentiation, indicating that thyroid hormone is one factor capable of regulating the transition between cell growth and differentiation in these cells. This terminal differentiation process is associated with induction of the cyclin/cyclin-dependent kinase inhibitors p21^{cip-1, waf-1} and p27^kip1, suggesting that thyroid hormone may regulate terminal differentiation in part by arresting cell cycle progression through induction of cyclin-dependent kinase inhibitors.

	Introduction

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TERMINAL DIFFERENTIATION of growth plate chondrocytes into hypertropic cells is an obligatory step in the endochondral ossification pathway that occurs during embryonic bone formation, longitudinal bone growth, and fracture healing. This terminal differentiation process is marked by a severalfold increase in cell volume, synthesis of type X collagen, and high levels of alkaline phosphatase activity (1). The net result of this developmental progression is an increase in length of the growing bone and mineralization of the surrounding cartilage matrix. This mineralized extracellular matrix of cartilage provides the scaffold for the deposition of new bone matrix by invading osteoblasts.

The fundamental importance of endochondral ossification in the growth, development, and repair of the skeleton has engendered a search for the molecular factors that regulate this critical pathway. Although many studies of the potential roles of systemic hormones and peptide growth factors in the regulation of endochondral ossification have been performed, these investigations have failed to define a clear mechanism of action for how many of these factors regulate this series of cellular events.

A prime example of this gap in our knowledge of how skeletal growth is regulated is thyroid hormone, which has been known for over 40 yr to be a critical regulator of skeletal maturation in vivo (2, 3, 4). The actions of thyroid hormone in enhancing longitudinal bone growth are among the most sensitive effects of this hormone. Deficiency of thyroid hormone in animals and humans results in delayed skeletal maturation, disorganization of the cartilage columns of the growth plates, and impaired differentiation of growth plate chondrocytes into hypertropic cells (4, 5). These abnormalities are often clinically manifested in hypothyroid children as severe growth retardation and mechanical failure of the growth plates of the hips (slipped capital femoral epiphyses) (6, 7).

Previous investigators have documented a positive role for thyroid hormone in regulating terminal differentiation of growth plate chondrocytes in vitro. This induction of the hypertropic chondrocyte phenotype by thyroid hormone has been observed in rat (8), chick (9, 10), and bovine (11) growth plate chondrocytes cultured in monolayer with or without serum, in rat epiphyseal pellet cultures with or without serum (12), and in organ cultures of rat femur (13) or porcine scapula (14).

Despite recent advances in the understanding of how systemic hormones and peptide growth factors regulate growth and differentiation in a number of target tissues, the molecular mechanisms by which thyroid hormone exerts these profound effects on bone growth and development have remained an enigma. It is generally accepted that cell division and cell differentiation are often mutually exclusive biological processes; therefore, a required step in terminal differentiation is withdrawal of a cell from the cell division cycle. A growing body of evidence suggests that systemic hormones and peptide growth factors may exert their effects on cell growth and differentiation in part through regulation of this cell division cycle (15, 16, 17).

We hypothesize that the cell cycle is a critical interface for integrating the input from the multiple local and systemic signaling molecules present in the growth plate, and that thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through controlling cell cycle progression at the G₁/S restriction point. Our results support this hypothesis by demonstrating that thyroid hormone regulates the critical transition between cell growth and cell differentiation in epiphyseal chondrocytes maintained under chemically defined, serum-free culture conditions, and that this induction of terminal differentiation is associated with up-regulation of the cyclin-dependent kinase (cdk) inhibitors p21^{cip-1, waf-1} and p27^kip1.

	Materials and Methods

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Chondrocyte isolation
Chondrocytes were isolated from the resting zone of the distal femoral growth cartilage of 1-day-old neonatal Sprague Dawley rats by collagenase digestion. The distal femora were surgically exposed, adherent soft tissues were stripped away, and the reserve zone of the epiphysis was removed by sharp dissection. The cartilage fragments containing the reserve zone chondrocytes were washed three times in PBS containing 1% penicillin/streptomycin and digested for 4 h in a 0.3% solution of collagenase P (Roche Molecular Biochemicals, Indianapolis, IN) in a shaking incubator at 37 C. The solution containing the isolated cells was filtered through 70-µm pore size mesh, and the cells were recovered by centrifugation at 200 x g at 4 C and resuspended in culture medium at a final concentration of 200,000 cells/ml.

Three-dimensional pellet culture
Chondrocytes were cultured as a three-dimensional cell pellet as previously described (12, 18, 19). Briefly, 1-ml aliquots containing 200,000 cells each were added to 15-ml conical polypropylene centrifuge tubes, and the cells were pelleted by centrifugation at 200 x g for 5 min at 4 C. The cultures were then maintained at 37 C in 95% room air and 5% CO₂ in a humidified incubator. Freshly isolated cells were allowed to recover for 3 days after collagenase digestion and cell pelleting before addition of thyroid hormone at the first medium change. Medium was then changed three times per week after the third day.

Pellets were maintained in growth medium consisting of DMEM/Ham’s F-12 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 50 µg/ml L-ascorbic acid phosphate (Wako Biochemicals, Osaka, Japan), 100 µg/ml sodium pyruvate (Life Technologies, Inc.), 1% (vol/vol) penicillin-streptomycin (Life Technologies, Inc.), and a defined media supplement (ITS+ Pre-Mix, Collaborative Biochemicals, Bedford, MA) yielding a final concentration of 6.25 µg/ml bovine insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenous acid, 1.25 mg/ml BSA, and 5.35 µg/ml linoleic acid. Control cultures were maintained in growth medium alone, whereas in the experimental cultures either L-T₄ (100 ng/ml) or T₃ (100 ng/ml; Sigma, St. Louis, MO) was added to the insulin-containing growth medium at the first medium change and readded with each change of the culture medium.

DNA content
The DNA content of the pellet cultures was determined on days 1, 2, 3, 5, 7, 14, and 21 after thyroid hormone treatment (20). Pellets were washed twice with cold PBS and homogenized in 1 ml homogenization buffer (0.05 M Na₂PO₄ and 2 M NaCl, pH 7.4). To elicit fluorescence, samples were treated with 0.1 µg/ml Hoechst 33258 dye, and absorbance at wavelengths of 365 and 460 nm was measured in a spectrophotometer. DNA content in the crude homogenates was then calculated based on a standard curve generated from known DNA concentrations. Statistical analysis was performed by two-way ANOVA for the variables time and treatment.

Northern blotting
Pooled pellets (20–40/group) were snap-frozen, and total cellular RNA was extracted by homogenization in 6 M guanidine HCl, followed by cesium chloride gradient centrifugation and sequential lithium chloride and lithium chloride/ethanol precipitation. Total cellular RNA (5 or 10 µg/lane) was fractionated by electrophoresis through 1% agarose gels, transferred to nylon membranes, and cross-linked to the membrane by exposure to UV light. Radiolabeling of complementary DNA (cDNA) probes with [³²P]deoxyCTP was performed using the random priming technique. Membranes were prehybridized, hybridized, and washed by the method of Church and Gilbert (21), thenexposed to radiographic film at -70 C using intensifying screens.

The following plasmids were used a sources of cDNA sequences: pSAM10 h, containing 600 bp of exon 3 and the 3'-untranslated region of the mouse type X collagen gene (22) (a gift from Suneel Apte, Cleveland Clinic Foundation, Cleveland, OH); pRAP54, which contains a 2.4-kb fragment of the rat alkaline phosphatase cDNA (23) (a gift from Mark Thiede, Pfizer, Inc., Groton, CT); pCRW 0.8, containing 0.8 kb of the rat p21^{cip-1, waf-1} cDNA (24) (a gift from Bert Vogelstein, The Johns Hopkins University, Baltimore, MD); pBSp27, containing a 0.9-kb DNA fragment encoding the open reading frame of the mouse p27^kip-1 cDNA (25) (a gift from Tony Hunter, The Salk Research Institute, San Diego, CA); pKS-mp15 and pKS-mp16, containing 1.3- and 0.8-kb fragments encoding full-length mouse p15^ink4B and p16^ink-4A cDNAs, respectively (26) (gifts from Charles J. Scherr, St. Jude Children’s Research Hospital, Memphis, TN), and a PstI fragment of the rat glyceraldehyde-3-phosphate dehydrogenase cDNA for assessment of equivalence of RNA loading.

Alkaline phosphatase activity
Pellets were homogenized in 2 ml ice-cold 0.15 M NaCl containing 3 mM NaHCO₃ (pH 7.4) with three 10-sec bursts using a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) and centrifuged at 10,000 x g for 30 min at 4 C. The supernatants were assayed for alkaline phosphatase activity in 0.1 M sodium barbital buffer (pH 9.3) using p-nitrophenyl phosphate as a substrate (27). The amount of protein contained in the enzyme extracts was determined by the Lowry method (28), and the enzyme activity was expressed as units of alkaline phosphatase activity per mg protein. One unit of alkaline phosphatase activity was defined as the enzyme activity that liberates 1 µmol p-nitrophenol at 37 C/mg protein/30 min. Statistical analysis of mean alkaline phosphatase values from triplicate cultures was performed by two-way ANOVA.

Western blotting
Cell lysates were prepared from five pellets per time point. The pellets were snap-frozen and pulverized under liquid nitrogen. The pulverized material was added to 250 µl ice-cold RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, and 150 mM NaCl, pH 7.4) containing 5 µl phenylmethylsulfonylfluoride (10 mg/ml), kept on ice for 30 min, and sonicated twice for 5 sec each time. Insoluble material was removed by centrifugation at 14,000 x g for 20 min at 4 C. After protein quantitation by the method of Lowry et al. (28), equal amounts of cell lysates were separated on 14% SDS-PAGE gels and transferred to nitrocellulose by electroblotting (0.8 mA/cm² for 90 min). Nonspecific binding was blocked by incubating the membrane in Blotto A (5% nonfat dry milk and 0.1% Tween-20 in PBS) for 1 h. The membrane was then incubated with primary antibody at a concentration of 1 µg/ml in Blotto for 1 h and washed twice for 10 min each time with 0.1% Tween-20 in PBS. Primary antibodies included anti-p21 [C-19 (catalog no. sc-397, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and SX118, (catalog no. 65951A, PharMingen, San Diego, CA)], anti-p27 (N-20, catalog no. sc-527; Santa Cruz Biotechnology, Inc.), anti-p15 (K-18, catalog no. sc-613, Santa Cruz Biotechnology, Inc.), and anti-p16 (M-156, catalog no. sc-1207, Santa Cruz Biotechnology, Inc.). Secondary antibody (antirabbit or antigoat IgG conjugated to horseradish peroxidase) was diluted 1:3,000 in Blotto and incubated with the membrane for 30 min, followed by three washes of 5 min each in 0.1% Tween-20 in PBS and a final 5-min wash with PBS alone.

Protein was visualized by incubating the membrane with chemiluminescence reagent (Renaissance, NEN Life Science Products, Boston, MA) for 1 min at room temperature and exposing the membrane to radiographic film for 5 min.

	Results

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Thyroid hormone regulates the transition between cell growth and cell differentiation in epiphyseal chondrocyte cell pellets
Chondrocyte pellet cultures maintained in chemically defined, serum-free growth medium increased DNA content from days 1–21 (Fig. 1). In pellets treated with thyroid hormone (100 ng/ml), DNA content also increased for the first 7 days after addition of the hormone; however, no further increase was observed over the remainder of the culture period. Two-way ANOVA demonstrated a significant effect of both time and treatment on DNA content (P < 0.001) as well as a significant interaction between these two variables (P < 0.001).

View larger version (14K): [in this window] [in a new window]   Figure 1. DNA content of pellet cultures of epiphyseal chondrocytes by thyroid hormone. Epiphyseal chondrocytes were isolated from reserve zone of the distal femoral epiphysis of 1-day-old neonatal Sprague Dawley rats by collagenase digestion on day -3 and allowed to recover in chemically defined medium for 3 days before the addition of T4 on day 0. DNA content was measured at each time point by fluorometry. Error bars represent the SEM.

View larger version (56K): [in this window] [in a new window]   Figure 2. Thyroid hormone stimulates expression of type X collagen mRNA and alkaline phosphatase mRNA. Northern analysis of total cellular RNA (5 µg/lane) extracted from epiphyseal chondrocyte pellet cultures treated with or without thyroid hormone and hybridized to a cDNA encoding type X collagen (A; 7-h exposure) and alkaline phosphatase (B; overnight exposure). Hybridization of glyceraldehyde-3-phosphate dehydrogenase mRNA was performed for normalization of loading. C, Control cultures demonstrated no type X collagen expression (data not shown) and very low levels of alkaline phosphatase expression (overnight exposure).

View larger version (17K): [in this window] [in a new window]   Figure 3. Thyroid hormone stimulates alkaline phosphatase enzymatic activity. Pellets treated with or without thyroid hormone were assayed for the presence of alkaline phosphatase enzymatic activity as measured by the conversion of para-nitrophenyl phosphate to para-nitrophenol. Activity is presented as units of para-nitrophenyl phosphate converted per mg protein/30 min. Error bars represent the SEM.

View larger version (56K): [in this window] [in a new window]   Figure 4. T3 is more potent than T4 in inducing markers of terminal differentiation. Northern analysis was performed of total cellular RNA (5 µg/lane) extracted from epiphyseal chondrocytes after treatment for 1–7 days with either T4 (100 ng/ml) or T3 (100 ng/ml) and hybridized to a cDNA probe encoding mouse type X collagen (overnight exposure).

Terminal differentiation of epiphyseal chondrocytes is associated with the induction of cdk inhibitors p21^{cip-1, waf-1} and p27^kip1
Pellets maintained in growth medium alone or growth medium containing T₃ (100 ng/ml) for 1–7 days were analyzed for expression of genes encoding the cyclin-dependent kinase inhibitor proteins p21^{cip-1, waf-1}, p27^kip-1, p15^ink4b, and p16^ink4a. Each experiment was performed on three separate occasions with similar results, and the figures are representative of the three experiments. Expression of p21^{cip-1, waf-1} mRNA increased on day 4 in pellets treated with thyroid hormone compared with controls, and this increase persisted on day 7 (Fig. 5A). In the control pellets, p21^{cip-1, waf-1} expression decreased as a function of time. Expression of p27^kip-1 mRNA was not appreciably affected by thyroid hormone treatment (Fig. 5B). Expression of p16^ink4a mRNA increased slightly in the thyroid hormone-treated pellets on days 4 and 7 compared with the controls (Fig. 5C). Expression of p15^ink4b mRNA by epiphyseal chondrocytes was not detectable by Northern hybridization of total RNA even after prolonged exposure of the autoradiographs (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 5. Thyroid hormone induces expression of the gene encoding the cdk inhibitor p21cip-1, waf-1. Northern analysis of total cellular RNA (10 µg/lane) extracted from epiphyseal chondrocyte cell pellets treated with or without thyroid hormone and hybridized to a cDNA probe encoding p21cip-1,waf-1 (A; overnight exposure), p27kip-1 (B; overnight exposure), and p16ink4a (3-day exposure).

View larger version (37K): [in this window] [in a new window]   Figure 6. Thyroid hormone increases levels of intracellular protein for the cdk inhibitor p21cip-1, waf-1, but not p27kip-1 or p16ink4a. Immunoblots of whole cell lysates from epiphyseal chondrocyte pellet cultures treated with or without thyroid hormone were performed using antibodies to p21cip-1, waf-1, p27kip-1, or p16ink4a.

	Discussion

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Numerous investigations have documented the presence and activity of a host of locally synthesized regulatory molecules in the growth plate that are capable of modulating cell growth and/or differentiation under various conditions. In addition, a number of systemically active hormonal regulators of growth plate chondrocyte function have been described. It is possible that a mechanism(s) exists to allow the growth plate chondrocyte to integrate the input from these various signaling molecules to determine whether the cell will enter another round of cell proliferation or to terminally differentiate into a hypertropic chondrocyte. We postulate that the integration of this signaling information may occur at least in part at the level of the cell division cycle.

The cyclin-cdk inhibitors described to date have been classified into two major groups: the cip-kip family (p21^cip-1,waf-1, p27^kip-1, and p57^kip-2) and the ink4 family (p15^ink4b, p16^ink4a, p18, and p19). One of these cyclin-cdk inhibitor proteins, p21^{cip-1, waf1}, is expressed at high levels in many cell types undergoing terminal differentiation in vivo, suggesting that arrest of cell cycle progression at the G₁/S boundary may be a common feature of terminally differentiating cells (15, 30, 31, 32, 33, 34). Although it is known that p21 is a universal cyclin-cdk inhibitor, the other related proteins appear to be more restricted in their action. However, the important functional differences between these proteins and the specific intracellular targets of their inhibitory action remain largely unidentified.

A growing body of evidence indicates that systemic hormones and peptide growth factors may exert their effects on cell growth and differentiation in part through regulation of the cell division cycle (35, 36, 37). Studies of breast carcinoma cells have revealed induction of cyclin D1 expression associated with changes in cell cycle progression by estrogens and progestins (38). Other studies of cell differentiation in nonskeletal tissues suggest a specific role for thyroid hormone in controlling cell cycle progression. For example, oligodendrocyte precursor cells fail to differentiate into oligodendrocytes in vitro unless thyroid hormone is present in the medium. The role of thyroid hormone in this model of cell differentiation is to induce the oligodendrocyte precursor cells to stop dividing and withdraw from the cell cycle (39). As systemic hormones such as thyroid hormone and estrogen are also known to be involved in regulation of the rate of skeletal growth, these observations suggest that control of cell cycle progression in growth plate chondrocytes may be a general mechanism of hormonal regulation of skeletal growth and maturation.

Certain peptide growth factors have also been shown to exert their effects on cell growth and differentiation through regulation of cell cycle progression (36, 40). For example, transforming growth factor-ß, which we have shown inhibits the differentiation of epiphyseal chondrocytes into hypertropic cells in our model (19), is now known to regulate cell cycle progression in other cell types through its effect on the cyclin-dependent kinase inhibitors p15^ink4B and p27^kip-1 (41, 42). Transforming growth factor-ß is also able to regulate promoter activity of the p21^{cip-1, waf-1} gene through the Sp1 transcription factor (43).

A recent report indicates that the expression of p21^{cip-1, waf-1} is elevated in tissues undergoing terminal differentiation in vivo, including skeletal muscle, skin, and nasal epithelium (33). Increased expression of p21^{cip-1, waf-1} has also been observed in a number of primary cell cultures and tumor cell lines undergoing differentiation in vitro, including primary keratinocytes (31), C2 myoblasts (15, 33), murine erythroleukemia cells (44), and HL-60 promyelocytic leukemia cells (32).

The most convincing evidence for a role of cyclin-cdk inhibitors in the process of cell differentiation is in the myelomonocytic cell line U937, which differentiates along the monocyte/macrophage pathway in response to 1,25-dihydroxyvitamin D₃. Using a strategy designed to identify early genes whose transcription is up-regulated by vitamin D₃ during differentiation, Liu et al. identified p21^{cip-1, waf-1} as one of the early genes transcriptionally up-regulated by vitamin D₃ (45). Forced overexpression of p21^{cip-1, waf-1} and/or p27^kip-1 in the absence of vitamin D₃ resulted in the expression of monocyte/macrophage-specific cell surface markers. Inducible expression of antisense p21 in these cells results in inhibition of vitamin D-induced differentiation as well as apoptosis (46). These results suggest that in some situations, transcriptional induction of cyclin-cdk inhibitors is sufficient to directly induce differentiation and may be required to prevent apoptosis.

Our data demonstrate that treatment of epiphyseal chondrocytes with thyroid hormone under chemically defined conditions results in the arrest of DNA synthesis and the onset of terminal differentiation, indicating that thyroid hormone is one factor capable of regulating the transition between cell growth and differentiation in these cells. This in vitro terminal differentiation process is associated with induction of the cyclin-cdk inhibitors p21^{cip-1, waf-1} and p27^kip1. These data are consistent with previous in vivo immunohistochemical and in situ hybridization data demonstrating increased p21^{cip-1, waf-1} levels in hypertropic chondrocytes of the growth plate (47). These results therefore suggest that thyroid hormone may regulate terminal differentiation in part by arresting cell cycle progression through induction of cyclin-dependent kinase inhibitors. Other possible targets for thyroid hormone regulation include paracrine factors in the growth plate, such as PTH-related peptide, Indian hedgehog, and members of the bone morphogenetic protein family of proteins and their antagonists.

These experiments do not answer the intriguing question of whether the induction of growth arrest alone is sufficient to cause terminal differentiation in this model system, or whether additional thyroid hormone-mediated signals are required for this to occur. Experiments in which transgenes encoding cdk inhibitors are overexpressed in epiphyseal chondrocytes in the absence of thyroid hormone will be necessary determine whether growth arrest alone is sufficient to cause terminal differentiation.

Received April 4, 2000.

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