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Keywords:

  • osteoporosis;
  • bone metastases;
  • anabolic;
  • Runx2;
  • valproate

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

HDIs are potential therapeutic agents for cancer and neurological diseases because of their abilities to alter gene expression, induce growth arrest or apoptosis of tumors cells, and stimulate differentiation. In this report, we show that several HDIs promote osteoblast maturation in vitro and in calvarial organ cultures.

Introduction: Histone deacetylase inhibitors (HDIs) are currently in phase I and II clinical trials as anticancer agents. Some HDIs are also commonly prescribed treatments for epilepsy and bipolar disorders. Although administered systemically, the effects of HDIs on osteoblasts and bone formation have not been extensively examined. In this study, we investigated the effect of histone deacetylase inhibition on osteoblast proliferation and differentiation.

Materials and Methods: MC3T3-E1 cells, calvarial-derived primary osteoblasts, and calvarial organ cultures were treated with various commercially available HDIs (trichostatin A [TSA], sodium butyrate [NaB], valproic acid [VPA], or MS-275). The effects of these inhibitors on cell proliferation, viability, cell cycle progression, Runx2 transcriptional activity, alkaline phosphatase production, and matrix mineralization were determined. Expression levels of osteoblast maturation genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in response to TSA were measured by quantitative PCR.

Results: Concentrations of HDIs that caused hyperacetylation of histone H3 induced transient increases in osteoblast proliferation and viability but did not alter cell cycle profiles. These concentrations of HDIs also increased the transcriptional activity of Runx2. TSA accelerated alkaline phosphatase production in MC3T3-E1 cells and calvarial organ cultures. In addition, TSA accelerated matrix mineralization and the expression of osteoblast genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in MC3T3-E1 cells.

Conclusions: These studies show that histone deacetylase activity regulates osteoblast differentiation and bone formation at least in part by enhancing Runx2-dependent transcriptional activation. Therefore, HDIs are a potentially new class of bone anabolic agents that may be useful in the treatment of diseases that are associated with bone loss such as osteoporosis and cancer.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

TRANSIENT POST-TRANSLATIONAL modification of the core histones facilitates chromatin remodeling and controls gene expression. Acetylation is one such reversible modification. Histone acetyltransferases (HATs) transfer acetyl groups to lysine residues on many proteins, including histones. Their recruitment to promoters facilitates RNA polymerase II activity and transcriptional activation. HATs are counteracted by histone deacetylases (HDACs), which remove acetyl groups from histones and promote chromatin condensation and transcriptional repression.(1–3) The 18 known HDACs are classified into three groups on the basis of their sequence homology to yeast molecules. Class I HDACs (1–3, −8, and −11) are homologous to the Saccharomyces cerevisiae RPD3 protein and are found in the nuclei of most mammalian cells.(4–6) Class II HDACs (4–7, 9, and 10) are homologous to the S. cerevisiae HDA1 protein, often shuttle between nuclear and cytoplasmic compartments, and usually exhibit tissue-specific expression patterns.(7–13) Class III HDACs (SIRT1–7) are homologous to the S. cerevisiae Sir2 protein and require NAD+ for deacetylase activity.(14,15)

Several classes of HDAC inhibitors (HDIs) have been identified including hydroxamic acids, cyclic peptides, butyrate, and benzamides.(16–19) HDIs block most class I and II HDACs and promote histone hyperacetylation and gene expression but have no effect on class III HDACs. In addition, HDIs induce cell cycle arrest, differentiation, and/or apoptosis of tumor cell lines in vitro and inhibit tumor growth in vivo.(17,20) Normal cells also undergo cell cycle arrest and differentiation in the presence of HDIs; however, they are less sensitive to HDI-mediated apoptosis because their cell cycle checkpoints are intact.(21) The tumor-selective effects of HDAC inhibitors make them promising anticancer agents and several are currently in phase I and II clinical trials for leukemias and solid tumors.(22) In addition to metastatic and primary cancers, HDIs hold promise as therapeutics for other diseases such as rheumatoid arthritis and cardiocyte hypertrophy because of their ability to alter gene expression and reprogram cellular activities.(23,24)

HDIs are administered systemically to patients; therefore, it is necessary to understand their effects on normal cells and the microenvironment surrounding malignant cells. Despite the plethora of data describing the consequences of HDI treatment on cancer cell lines and some nontransformed cell lines, little is known about how HDIs affect osseous cells. In 1993, Iwami and Moriyama(25) showed that sodium butyrate (NaB), recognized at the time only as a differentiation agent but not as a weak HDI, induced alkaline phosphatase (ALP) expression in the MC3T3-E1 cell line. More recently, Sakata et al.(26) showed that trichostatin A (TSA) increased osteopontin expression in C3H10T1/2 pre-osteoblast cells. Neither of these studies examined the effects of HDIs on osteoblast proliferation, maturation, or mineralization.

We recently showed that RNAi-mediated suppression of HDAC3 in MC3T3-E1 osteoblasts accelerates mineralization and expression of osteocalcin, osteopontin, and bone sialoprotein.(27) Because HDAC3 interacts with Runx2, these effects are at least partially caused by the relief of Runx2 repression. In this study, we aimed to determine if chemical inhibitors of HDACs, some of which are approved to treat neurological diseases or are in clinical cancer trials, induce similar effects on osteoblast gene expression, proliferation, and differentiation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Cell culture

MC3T3-E1 cells were maintained and differentiated in MEM in the presence or absence of osteogenic stimuli (i.e., 50 μg/ml ascorbic acid and 10 mM β-glycerol phosphate) as previously reported.(27) During differentiation assays, HDIs were added at days 0 and 3. Primary calvarial osteoblasts were isolated as previously described.(28,29) Briefly, calvaria from newborn CD1 mice were collected and sequentially rinsed in Hank's balanced salt solution (HBSS; Invitrogen, Carlsbad, CA, USA) and serum-free MEM (Invitrogen). Calvaria were digested into a single cell suspension in serum-free α-MEM containing 2 mg/ml collagenase and 0.25% trypsin. Cells were washed and plated at 2 × 105 cells/10-cm plate. HDIs were added to MC3T3-E1 or primary osteoblasts at various concentrations for the indicated times. TSA, NaB, and VPA were purchased from Sigma (St Louis, MO, USA). MS-275 was obtained from Calbiochem (San Diego, CA, USA).

Antibodies

The rabbit polyclonal antibodies, anti-acetyl-lysine9/14 (K9/K14) histone H3 and anti-histone H3, were purchased from Upstate (Lake Placid, NY, USA). The monoclonal anti-β-catenin antibody was obtained from BD Biosciences Transduction Laboratories (Lexington, KY, USA).

Immunoblots

MC3T3-E1 cells were lysed with buffer containing 20 mM Tris-HCL, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and protease inhibitors (Mini Complete protease inhibitor cocktail tablets from Roche Applied Science, Indianapolis, IN, USA). Primary osteoblasts were lysed in a solution of 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and protease inhibitors. Proteins extracts were resolved with SDS-15% PAGE, transferred to Immobilon P (Millipore, Bedford, MA, USA) and immunoblotted with the indicated antibodies.

ALP activity assays

MC3T3-E1 cells were washed three times in PBS and lysed with 0.2% NP-40 and 1 mM MgCl2. After sonication, the lysates were spun at 3000 rpm for 15 minutes at 4°C. Supernatants were added to a reaction solution containing 0.6 M 2-amino-2-methyl-1-propanol (Sigma), 2.4 mM MgCl2, and 9.6 mM p-nitrophenyl phosphate (Sigma). After incubation at 37°C for 30 minutes, reactions were stopped with 2 N NaOH, and the absorbance was read at 410 nm. ALP activity was normalized to protein content. Protein concentration was determined using the DC Protein Assay system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Calvarial organ culture lysates were spun at 14,000 rpm for 5 minutes at 4°C. Supernatants were added to a reaction solution containing 2 M diethanolamine, 1 mM MgCl2, 20 mM l-homoarginine, and 85.1 mM p-nitrophenyl phosphate. The reactions were read kinetically at 405 nm. The change in absorbance per minute over a 5-minute period was determined.

Mineralization assays

Matrix mineralization was quantified as previously described.(30) Briefly, differentiating MC3T3-E1 cells were transferred into osseous differentiation media containing 1 μCi/ml45CaCl2. At the indicated time-points, the cells were washed with PBS, harvested in 10 mM Tris-Cl, pH 7.4, and mixed with 30% trichloroacetic acid (Sigma). After storage at 4°C overnight, the lysates were vortexed and centrifuged at 2000 rpm for 10 minutes. Radioactive isotope incorporation was measured by liquid scintillation counting.45Ca accumulation was normalized to protein content. Protein concentration was determined with the DC Protein Assay system (Bio-Rad).

Cytotoxicity assays

Cells were cultured in 96-well plates at a concentration of 5000 cells/well. After 3 days in the presence or absence of the indicated HDI, cytotoxicity was determined using the CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay (Promega, Madison, WI, USA) system according to the manufacturer's instructions. The absorbance of formazan was measured at 490 nm on a Versamax plate reader with Softmax Pro software. Each measurement was made in quadruplicate, and percent viability was calculated relative to the untreated samples.

Cell cycle analysis

MC3T3-E1 cells were trypsinized, collected by centrifugation (2500 rpm, 4°C, 15 minutes) in PBS, fixed in 100% ethanol, and stored at −20°C for at least 30 minutes. The cells were collected by centrifugation (2500 rpm, 4°C, 15 minutes) and resuspended in HBSS containing 50 μg/ml propidium iodide and 100 μg/ml ribonuclease A. After a 30-minute incubation at room temperature, the DNA content of the cells was measured with a FACSCalibur (BD Biosciences, San Jose, CA, USA) flow cytometer. The data were analyzed using ModFit software (Verity Software House, Topsham, ME, USA).

cDNA synthesis and real-time quantitative PCR

Total RNA was isolated from MC3T3-E1 cells with Trizol reagent (Invitrogen). Total RNA (1 μg) was reverse transcribed to cDNA with the Invitrogen Superscript Kit. The amplification of osteocalcin, osteopontin, bone sialoprotein, and actin messages was performed as previously reported.(27) For detection of type I collagen, cDNA was reversed transcribed and amplified in a Lightcycler (Roche Diagnostics) with the Qiagen Quantitect SYBR Green RT-PCR kit using the following primers: type I collagen (5′-CCACGCATGAGCCGAAGCTAACCCC-3′, 5′-CTTCCCCATCATCTCCATTCTT-3′). Quantification and normalization to actin amplicons were performed as previously described.(27)

Transcription assays

MC3T3-E1 cells were transiently transfected with the murine osteocalcin gene 2 (mOG2)-Luc reporter and pRL-TK (Renilla luciferase). Luciferase activity was measured 40 h after transfection with the Dual Luciferase Assay System (Promega). Luciferase values were normalized with Renilla luciferase values to control for transfection efficiency. The fold changes in activation were calculated relative to samples transfected with mOG2-Luc alone. TSA, MS-275, VPA, and NaB were added 24 h after transfection.

Murine calvarial organ cultures

Calvarial organ cultures were performed as previously described.(31,32) Briefly, calvaria were isolated from 4-day-old CD1 mice and divided along the sagittal suture. Each half was placed on a stainless steel grid in a 12-well tissue culture plate containing BGJ medium (Invitrogen) supplemented with 0.1% BSA, 50 U/ml penicillin, 50 μg/ml streptomycin, and 50 μg/ml ascorbic acid. One-half of each pair was placed into medium-containing vehicle (ethanol for TSA, dimethyl sulfoxide (DMSO) for MS-275, PBS for NaB), whereas the other one-half from the same animal was cultured in medium containing the appropriate HDI (TSA, MS-275, NaB). After 24 h, the media were collected, and ALP activity was measured as described above.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

HDIs and histone acetylation in osteoblasts

We previously reported that HDAC3 suppression accelerates matrix mineralization and the expression of osteopontin, bone sialoprotein, and osteocalcin during osteoblast differentiation.(27) To determine how general inhibition of HDAC activity affects osteoblasts, we incubated MC3T3-E1 cells and primary osteoblasts with several commercially available HDIs. TSA and NaB caused hyperacetylation of histone H3 at lysines 9 and 14 in MC3T3-E1 cells. Histone H3 acetylation was evident with as little as 5 nM TSA and increased in a manner directly related to TSA concentration (Fig. 1A). Histones H2A, H2B, and H4 were also hyperacetylated (data not shown). TSA (10 nM) induced acetylation within 1 h. This acetylation was stable for at least 26 h (Fig. 1B). Nanomolar concentrations of TSA also induced hyperacetylation of histone H3 in calvarial-derived primary osteoblasts (Fig. 1D). NaB is a less potent HDI, and as expected, higher concentrations of it were needed to detect histone H3 acetylation in MC3T3-E1 cells. Peak acetylation occurred with 0.5 mM NaB (Fig. 1C). A fourth HDI, MS-275, induced acetylation of histone H3 at 500 nM in primary osteoblasts (Fig. 1E). These data reveal the minimal concentrations of HDIs that are needed for detection of hyperacetylated histones in both MC3T3-E1 cells and primary osteoblasts.

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Figure FIG. 1.. HDIs cause hyperacetylation of histone H3 in both MC3T3-E1 and primary osteoblasts. MC3T3-E1 or primary osteoblasts were treated with HDIs at the indicated concentrations. Lysates were collected 16 h after treatment unless otherwise specified. Immunoblots were performed using anti-acetyl histone H3 K9/K14, anti-histone H3 K9 or β-catenin antibodies. (A) MC3T3-E1 cells incubated with TSA. (B) MC3T3-E1 cultured with 10 nM TSA for the indicated times. (C) MC3T3-E1 cells cultured with NaB. (D) Primary osteoblasts treated with TSA. (E) Primary osteoblasts incubated with MS-275.

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Effects of HDIs on osteoblast viability

HDIs cause growth arrest in tumor cells and some normal fibroblasts and melanocytes.(17,20,21) We studied the effects of various HDIs on MC3T3-E1 and primary osteoblasts. HDIs induced morphological changes in MC3T3-E1 cells that became more severe with increasing concentrations of HDIs. In subconfluent conditions, MC3T3-E1 cells have an elongated, fibroblast-like appearance (Fig. 2). When exposed to HDIs (TSA, NaB, VPA) at concentrations that induce H3 hyperacetylation (Fig. 1), cells became flatter and spread across a larger surface area (Fig. 2). This phenotype became more prominent as higher concentrations of the HDIs were added to the cells. Similar results were observed with primary osteoblasts (data not shown).

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Figure FIG. 2.. HDIs cause morphological changes in MC3T3-E1 cells. MC3T3-E1 cells were incubated with the indicated HDIs for 16 h and analyzed by phase-contrast microscopy. (A) No HDI. (B) TSA. (C) NaB. (D) VPA.

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Despite morphological changes, cell viability was only lost with high concentrations of select inhibitors. MC3T3-E1 cells incubated with 5–50 nM of TSA were nearly as viable as control cells 1 day after initial exposure to the drug (data not shown). As shown in Fig. 1, these concentrations of TSA were sufficient to cause hyperacetylation of histones in these cells. Only higher concentrations of TSA, 100 and 1000 nM, reduced cell viability to 86.8% and 74.5%, respectively, after 1 day (data not shown). After 3 days of exposure, 5 nM TSA still had little effect on cell viability (92.2%), whereas 10–50 nM TSA reduced cell viability to 75.3–70.9% (Fig. 3A). Higher concentrations of TSA, 100 and 1000 nM, reduced viability to 55.8% and 51.0%, respectively, after 3 days. Similar effects were seen in primary osteoblasts cultures containing TSA (Fig. 4A). Thus, 5–50 nM TSA induced minimal cytotoxicity to MC3T3-E1 cells and primary osteoblasts.

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Figure FIG. 3.. HDAC inhibition does not reduce MC3T3-E1 viability. MC3T3 cells were incubated for 3 days with increasing concentrations of the indicated HDI. Viability was measured with the CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay system (Promega) according to the manufacturer's instructions. (A) TSA. (B) MS-275. (C) NaB. (D) VPA.

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Figure FIG. 4.. HDAC inhibition does not reduce primary cell viability. Primary calvarial cells were cultured for 3 days with increasing concentrations of the indicated HDI. Viability was measured with the CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay system (Promega) according to the manufacturer's instructions. (A) TSA. (B) MS-275. (C) NaB. (D) VPA.

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Interestingly, the three other HDIs that we tested enhanced the viability of primary osteoblasts and MC3T3-E1 cells. MS-275 (500 nM) stimulated the proliferation of MC3T3-E1 cells and primary osteoblasts after 3 days to 326.9% and 204.1%, respectively (Figs. 3B and 4B). NaB and VPA had similar effects in MC3T3-E1 cells because greater viability was observed after 3 days in cultures containing 0.1–50 μM of NaB or VPA (Figs. 3C and 3D). Higher concentrations of MS-275, NaB or VPA caused considerable cell death and thus a decrease in proliferation rate. Similarly, in primary osteoblasts, low concentrations of NaB or VPA (0.1–50 μM) augmented cell proliferation, although to a much lesser extent that what was observed in MC3T3-E1 cultures, and higher concentrations of NaB or VPA resulted in increased cytotoxicity (Figs. 4C and 4D). In summary, low concentrations of HDIs did not reduce cell viability in either MC3T3-E1 cells or primary osteoblasts but in some cases enhanced proliferation.

HDIs arrest tumor cell lines in G1 or G2 phase of the cell cycle. To determine if HDIs affect the cell cycle of osseous cells, we treated asynchronously growing MC3T3-E1 pre-osteoblasts with various concentrations of TSA or MS-275. No drastic changes in the cell cycle profiles were observed in cells cultured with 10–50 nM TSA (Table 1). However, high concentrations of TSA (1 μM) and MS-275 (10 μM) caused G1 arrest and considerable cell death. These results show that low concentrations of HDIs have no adverse effects on DNA synthesis and proliferation of osteoblasts.

Table Table 1.. Cell Cycle Profiles of MC3T3-E1 Cells Cultured With TSA or MS-275
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Effects of HDIs on osteoblast maturation

To determine the functional effects of HDIs on osteoblast maturation, a general class I and II HDI, TSA, was added to MC3T3-E1 cell cultures. TSA was chosen because it neither enhanced MC3T3-E1 cell proliferation nor adversely affected cell viability when used at nanomolar concentrations that were sufficient to induce H3-K9 acetylation. MC3T3-E1 cells were cultured in the presence of ascorbic acid and β-glycerol phosphate for 18 days. TSA (20 nM) was added to the differentiation media on days 0 and 3. ALP activity, an early marker of osteoblast differentiation, was measured on days 0, 3, 6, and 10. Cells incubated with 20 nM TSA produced 3-fold more ALP after 3 days than control cells (Fig. 5A); however, after 6 days, control and TSA-treated cells had similar levels of ALP activity. Matrix mineralization, a late marker of osteoblast maturation, was measured using a calcium incorporation assay. Approximately six times more calcium was incorporated into TSA-treated cultures on day 16 (Fig. 5B). This coincided with an increase in the average number of mineralized nodules per well. Untreated cell cultures contained 5 nodules per well, whereas the treated cultures harbored 31 nodules per well (data not shown). After culturing in the presence of45CaCl2 for 48 h, the treated cells incorporated ∼7-fold more calcium than the untreated cells (Fig. 5B). Protein concentrations in untreated and treated samples were similar at 20 and 48 h (data not shown).

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Figure FIG. 5.. TSA accelerates maturation of MC3T3-E1 cell cultures. (A) TSA transiently increases ALP activity. MC3T3-E1 lysates were collected on the indicated days, and ALP activity was measured. Open boxes represent control cells, and filled boxes represent cells cultured with 20 nM TSA. (B) TSA accelerates matrix mineralization.45CaCl2 was added to differentiating MC3T3-E1 cell cultures 15 days into the differentiation assay. Lysates were collected on days 16 (20-h lysate) and 17 (48-h lysate). White bars represent control cells, and black bars represent cells cultured with 20 nM TSA. (C-F) TSA accelerates the expression of type I collagen, osteopontin, bone sialoprotein, and osteocalcin. Real-time PCR was performed on cDNA that was isolated from differentiating MC3T3-E1 cells at the indicated time. Cells were incubated with either vehicle (ethanol) or 20 nM TSA for the first 6 days of the culture. White bars represent control cells, and black bars represent cells cultured with 20 nM TSA.

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We next analyzed the effects of TSA on the expression of osteoblast-specific genes that are induced during differentiation. RNA was collected from the cells at the indicated time-points, and quantitative PCRs were performed. At each time-point examined, type I collagen and bone sialoprotein were expressed at higher levels in the TSA-treated cells (Figs. 5C and 5E). Osteopontin was expressed at similar levels early in the differentiation protocol; however, at day 10, TSA-treated cells expressed ∼2.7-fold more osteopontin than untreated cells (Fig. 5D). A similar pattern was observed at day 16, when an ∼4.9-fold increase in the amount of osteopontin expressed in TSA-treated cells was observed. Osteocalcin levels were similar at the beginning of the differentiation protocol but peaked earlier in TSA-treated cells. At day 10, there was an ∼184-fold increase in osteocalcin levels in TSA-treated cells. By days 13 and 16, the untreated cells expressed more osteocalcin than the treated cells (Fig. 5F). These data show that suppression of HDAC activity early in osteoblast differentiation can accelerate the differentiation process.

Effects of HDIs on calvarial organ cultures

To determine the effect of HDIs on the maturation of osseous tissues, we cultured calvaria from 4-day-old mice in the presence or absence of TSA, MS-275, or NaB. After dissection, the calvaria were divided along the sagittal suture. One-half of each calvarium was cultured in the presence of an HDI, whereas the other one-half was cultured in medium containing vehicle only. After 24 h, the organ culture media were collected and tested for ALP activity. All four calvaria cultured with either 10 nM TSA, 20 nM TSA, or 500 nM MS-275 produced more ALP than their matched controls (Figs. 6A-6C). Three of four calvaria incubated with 500 nM NaB secreted more ALP than the matched untreated controls (Fig. 6D). On average, the amount of ALP produced by each set of treated calvaria was higher than the corresponding untreated controls. These results show that HDIs promote osteoblast maturation in calvarial tissues.

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Figure FIG. 6.. HDIs increase ALP activity ex vivo. Calvaria were dissected and divided along the sagittal suture. One-half of each calvarium was cultured in vehicle and the other one-half was incubated with the indicated HDI. After 24 h, media were collected and analyzed for ALP activity. The matching symbols in the control and HDI columns represent one mouse. Black lines represent average ALP production from the four animals in each set. (A) TSA (10 nM) or ethanol (vehicle). (B) TSA (20 nM) or ethanol (vehicle). (C) MS-275 (500 nM) or DMSO (vehicle). (D) NaB (500 nM) or PBS (vehicle).

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Effects of HDIs on Runx2 activity

In an effort to identify mechanisms by which HDIs enhance bone maturation, we focused on Runx2, a transcription factor necessary for osteoblast differentiation. We previously reported that HDAC3 interacts with Runx2 and represses Runx2-dependent transcriptional activity.(27) To determine if Runx2 transcriptional activity is upregulated in response to HDI treatment, TSA, MS-275, VPA, and NaB were added to transcription assays. MC3T3-E1 cells were transiently co-transfected with a Runx2 expression plasmid and two reporter plasmids, mOG2-Luc and pRL-TK. HDIs were added 24 h after transfection. Similar to our previously published results, TSA enhanced Runx2 transcriptional activity by 7.7- (10 nM) and 14-fold (20 nM; Fig. 7A).(27) MS-275, NaB, and VPA also increased Runx2 transcriptional activity from 5- to 12-fold (Figs. 7B-7D). These data suggest that Runx2 activity is enhanced with HDI treatment and reveal one mechanism for HDI-accelerated osteoblast differentiation.

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Figure FIG. 7.. HDIs increase Runx2 transcriptional activity. MC3T3-E1 cells were transfected with reporter plasmids, mOG2-Luc (0.2 μg) and pRL-TK (50 ng), and a mammalian expression vector for Runx2 (MASNS isoform; 0.5 μg). HDIs were added at the indicated concentrations 24 h after transfection. Luciferase activity was determined 40 h after transfection. (A) TSA. (B) MS-275. (C) VPA. (D) NaB.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

HDIs are promising anticancer agents because of their ability to promote terminal differentiation and/or induce growth arrest in numerous tumor cells; however, their effects on many normal cells types, including osteoblasts, are poorly understood.(33) It is important to discern their activities in normal tissues because these inhibitors are administered systemically. In this study, we investigated the effect of HDIs on osseous cells and tissues, specifically MC3T3-E1 cells, primary osteoblasts, and mouse calvaria. Inhibition of HDAC activity with a variety of HDIs did not induce growth arrest in osseous cells. However, HDAC inhibition accelerated in vitro osteoblast maturation and mineralization as measured by ALP production, calcium incorporation, and the expression of type I collagen, osteopontin, bone sialoprotein, and osteocalcin. In calvarial organ cultures, HDIs also increased ALP activity. In addition, HDIs increased Runx2-dependent activation of the osteocalcin promoter. We conclude that HDIs promote osteoblast maturation and the expression of osteoblast specific genes, at least in part, through upregulation of Runx2 activity.

Osteoblasts express multiple HDACs, which are likely to have specific roles in regulating osteoblast maturation. We previously reported that HDAC3 and HDAC6 are expressed in osteoblasts.(7,27) HDAC4 was detected in some, but not all osteoblast cell lines.(27,34,35) We have also observed HDAC1, HDAC2, HDAC5, HDAC7, and HDAC9 in osteoblasts by immunoblotting and/or PCR (unpublished data). In this study, the effects of blocking the enzymatic activity of most if not all of these HDACs during bone maturation were addressed. TSA was chosen for the maturation assays with MC3T3-E1 cells because it inhibits all class I and II HDACs,(36) is effective at low concentrations (5–20 nM), and is readily available from commercial sources. In performing these assays, we found that culturing MC3T3-E1 or primary osteoblasts with TSA at concentrations >20 nM or for >3 days was toxic to the cells. However, TSA only needed to be added twice (at days 0 and 3) to accelerate the appearance of early and late markers of osteoblast maturation. Type I collagen and bone sialoprotein transcript levels were consistently higher in cells cultured in the presence of TSA at all time-points examined. Interestingly, osteopontin and osteocalcin mRNA was not elevated in TSA-treated samples until day 10. Osteopontin remained elevated in TSA-treated samples on days 13 and 16, but osteocalcin levels were lower in TSA-treated samples at these time-points. The mechanisms responsible for these gene expression patterns are unknown but likely depend on promoter structure, nucleosomal organization and the temporal expression, activity, and/or recruitment of gene-specific transcription factors (e.g., Dlx5, Msx2, ATF4, Osterix, Smads) and co-regulatory proteins to the promoters. Examining how HDIs affect the organization of these and other genes will be important to fully understand how these chemical inhibitors promote osteoblast gene expression. Furthermore, HDIs likely affect the activities of many transcription factors and the expression of hundreds of genes in osteoblasts. Their ability to accelerate the appearance of multiple osteoblast differentiation marker genes indicates that they epigenetically reprogram the cell in such a way as to facilitate maturation.

The observation that HDIs accelerate osteoblast maturation was not unexpected. HDIs accelerate differentiation of many normal cell types. For example, VPA induces neuronal differentiation,(37) and NaB facilitates mammary epithelial cell differentiation.(38) Previous studies hinted that HDIs also stimulate osteoblast maturation. NaB-induced ALP production by MC3T3-E1 cells and TSA increased osteopontin expression in C3H10T1/2 pre-osteoblast cells.(25,26) Moreover, we previously showed that HDAC3 and HDAC6 interact with Runx2, a master osteoblast gene product, and repress its ability to transactivate the osteocalcin promoter.(7,27) HDIs relieved this repression and enhanced Runx2 activation of the osteocalcin promoter in osseous cells.(27) Our current data with additional HDIs agree with these results, but also show that HDIs promote matrix mineralization of osseous cultures and maturation of calvarial tissues. The underlying mechanism promoting osteoblast maturation on HDI treatment likely involves altering many gene transcription pathways including Runx2. Another possible mechanism is through activation of the canonical Wnt signaling pathway, which is important for osteoblast differentiation(39) and can be positively influenced by acetylation in a number of ways, including facilitating the nuclear localization of β-catenin.(40,41) We, however, did not observe an affect of TSA treatment on β-catenin localization in MC3T3-E1 cells (data not shown).

The four HDIs that we tested have varying structures, specificities, and potencies (ranging from nanomolar to millimolar concentrations). TSA is regarded as a general class I and II HDAC inhibitor, but VPA does not inhibit HDAC6 or HDAC10,(42) and MS-275 does not inhibit HDAC8.(43) Importantly, none of the HDIs induced osteoblast cytotoxicity when used at the minimal concentrations needed to detect acetylated histone H3, and some even enhanced cell proliferation. The ability of MS-275, NaB, and VPA to enhance osteoblast proliferation, whereas TSA did not, may involve the specificity of each inhibitor. At the present time, small molecule inhibitors that actively block specific HDACs are not available. We and others have used RNA interference to suppress specific HDAC molecules in an attempt to examine their roles in osteoblast maturation. In our previous study, specific suppression of one HDAC, HDAC3, resulted in the acceleration of mineralization and the expression of osteopontin, bone sialoprotein, and osteocalcin without affecting cell proliferation. Interestingly, HDAC3 suppression did not enhance ALP activity.(27) Our observation in this report that several HDIs enhance ALP expression suggests that HDACs other than HDAC3 must participate directly or indirectly in the regulation of this gene. Recently, Kang et al.(35) showed that suppression of either HDAC4 or HDAC5 in ROS 17/2.8 or caIB 2T3 cells relieved TGFβ-mediated and SMAD3-dependent repression of the osteocalcin promoter and mineralization. Together, these studies indicate that targeted suppression of individual HDACs by RNA interference is sufficient to accelerate various aspects of osteoblast maturation. Additional studies will be necessary to distinguish the roles of particular HDACs during osteoblast differentiation.

Our results show that HDIs positively affect osteoblast maturation in short-term assays and suggest that HDIs may be useful for promoting bone formation. It is also important to consider the long-term effects of HDI administration on bone strength. VPA has been used as an anticonvulsant and mood-stabilizing drug in epileptic and bipolar patients since 1962. Recent studies showed that long-term administration (at least 6 months) of VPA is associated with decreased BMD, osteopenia, and osteoporosis in children and adults.(44–46) Consequently, these individuals have an increased fracture risk.(47) The changes in BMD do not seem to be caused by VPA, but rather are attributed to decreased physical activity levels, vitamin D deficiency, and secondary hyperparathyroidism.(46,48) Nevertheless, additional studies examining the long-term effects of HDIs, as well as the consequences of sustained HDI administration on bone formation, are needed.

Several HDIs are now in clinical trials to treat hematopoietic and solid tumors.(22) Many aggressive cancers, including those of the breast, prostate, and lung, metastasize to bones and alter bone formation and/or bone resorption processes.(49) Fractures, pain, and decreased bone strength associated with bone metastases greatly diminish the quality of life of cancer patients. HDIs induce tumor cell cycle arrest and increase the radiosensitivity of tumors.(50–53) Our data show that HDIs enhance bone formation. Others reported that HDIs block bone resorption by inhibiting osteoclastogenesis of rat bone marrow cells.(54) The effects of HDIs on osteoblast and osteoclast maturation in the context of the bone microenvironment and tumor microenvironment remain to be determined. It is possible that signals from surrounding cells and the microenvironment will abrogate or otherwise alter any effects seen in the culture dish. Nevertheless, initial results suggest that HDIs may effectively block metastatic cancer growth and cancer-associated bone disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank Paul Marker and Sheri Kuslak for generous gifts of reagents and Felix Rosenow for insightful discussions. We are grateful for the assistance provided by the Flow Cytometry Core Facility in The Cancer Center of the University of Minnesota. The National Institute of Arthritis, Musculoskeletal and Skin Diseases (AR48147 and AR050074), the National Cancer Institute (CA09138), and The V Foundation for Cancer Research supported this work.

REFERENCES

  1. Top of page
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  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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