Matrix-metalloproteinase-13 (MMP-13) has been shown to be an important protease in inflammatory and neoplastic conditions of the skeletal system. In particular, the stromal cells of giant cell tumor of bone (GCT) express very high levels of MMP-13 in response to the cytokine-rich environment of the tumor. We have previously shown that MMP-13 expression in these cells is regulated, at least in part, by the RUNX2 transcription factor. In the current study, we identify the expression of the c-Fos and c-Jun elements of the AP-1 transcription factor in these cells by protein screening assays and real-time PCR. We then used siRNA gene knockdown to determine that these elements, in particular c-Jun, are upstream regulators of MMP-13 expression and activity in GCT stromal cells. We conclude that there was no synergy found between RUNX2 and AP-1 in the regulation of the MMP13 expression and that these transcription factors may be independently regulated in these cells.
Bone resorption involves dissolution of the nonorganic component of bone, specifically hydroxyapatite, followed by degradation of the collagenous component composed mostly of type-I collagen [
Giant cell tumor of bone (GCT) is an aggressive and highly osteolytic bone tumor that is characterized by rapid bone destruction. The cellular elements of GCT include both osteoclast-like giant cells and osteoblast-like stromal cells [
The Runx2 binding site OSE2 has been found to colocalize with the activator protein 1 (AP-1) transcription factor binding site (not limited to the c-Fos and c-Jun elements) in the promoter region of MMP-13 in human cells [
The use of all patient-derived material was approved by our institution’s Research Ethics Board, and patient informed consent was obtained individually. The diagnosis of GCT of bone was established by biopsy prior to surgical excision. Specimens were obtained at the time of surgery from patients undergoing tumor resection, and a bone pathologist verified the diagnosis of GCT postoperatively. Tissue samples from four cases of GCT of bone were used in this study, and all experiments were performed in triplicate or as otherwise stated for all four bone tumors.
We established primary cell cultures of GCT stromal tumor cells from fresh GCT tissue. The specimens were freshly minced in Dulbecco’s Modified Eagle Medium (D-MEM, Gibco, Burlington, ON) producing a cell suspension with small fragments of tissue. The resultant suspension was passed through a 20-gauge needle prior to seeding in cell culture flasks with D-MEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100
GCT stromal cells were grown to
Mesenchymal stromal cells of GCT were trypsinized and transfected with Runx2, c-Jun, and c-Fos small interfering RNAs (siRNAs) via electroporation. Stromal cells of GCT were washed and resuspended in Opti-MEM I reduced-serum medium (Gibco). Subsequently, cell suspension was mixed with either 200 nM of ON-TARGETplus SMARTpool Runx2 siRNA (Thermo Scientific-Dharmacon), Stealth c-Fos and c-Jun siRNA (Invitrogen), a positive Silencer siRNA control against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or a nonspecific negative control no. 1 (Ambion Inc.). Stromal cells with siRNA mixture were electroporated using the Gene Pulser II electroporation apparatus (Bio-Rad Laboratories) under a single-pulse protocol with optimized combinations of voltage and capacitance. Then, cells were plated in cell culture flasks with supplemented D-MEM. At 48 hours after the transfection, cells were harvested for Runx2 mRNA. Ribosomal protein S18 (RPS18) was selected among other housekeeping genes for normalization in real-time PCR analysis since GAPDH has been used as the positive siRNA control. The viability of stromal cells after transfection was evaluated by hemocytometry.
Total RNA was isolated from GCT stromal cells using the RNeasy Mini Kit (Qiagen, ON) as optimized in our lab. To ensure complete removal of contaminating genomic DNA prior to first-strand synthesis, RNase-free DNase I treatment was applied on the RNeasy column during total RNA isolation. Single-stranded complementary DNA (cDNA) was synthesized from 1.0
The expression of GAPDH, Runx2, MMP-13, c-Fos, and c-Jun in cells treated with various siRNAs were analyzed using real-time RT-PCR. In brief, real-time PCR analysis was performed on cDNA synthesized from GCT stromal total RNA using the MiniOpticon Real-Time PCR Detection System with the iQ SYBR Green Supermix (Bio-Rad Laboratories, ON) according to the manufacturer’s instructions. Cycling consisted of 40 cycles of 15 s at 95°C, 30s at 58°C, and 30 s at 72°C, operated with the Opticon Monitor software v3.1. PCR experiments were performed in triplicate and included negative notemplate controls. Primer pairs (Table
Human primer sequences specially designed for real-time RT-polymerase chain reaction (PCR) amplification.
Gene | Forward/Reverse | Primer sequence | Accession no. | Size of product (bp) | Melting temperature (°C) |
---|---|---|---|---|---|
c-Fos | F | 5' AGA ATC CGA AGG GAA AGG AA 3' | NM_005252 | 150 | 63.6 |
R | 5' CTT CTC CTT CAG CAG GTT GG 3' | 63.8 | |||
c-Jun | F | 5' CAG GTG GCA CAG CTT AAA CA 3' | NM_002228 | 80 | 63.8 |
R | 5' GTT TGC AAC TGC TGC GTT AG 3' | 63.5 | |||
MMP-13 | F | 5' CTT CCC AAC CGT ATT GAT GC 3' | NM_002427 | 143 | 64.1 |
R | 5' TTT GGA AGA CCC AGT TCA GA 3' | 62.2 | |||
Runx2 | F | 5' TCT GGC CTT CCA CTC TCA GT 3' | NM_004348 | 142 | 64.0 |
R | 5' AAG GTG GCT GGA TAG TGC AT 3' | 63.4 | |||
RPS18 | F | 5' GAT GGG CGG CGG AAA ATA G 3' | NM_022551 | 165 | 68.4 |
R | 5' GCG TGG ATT CTG CAT AAT GGT 3' | 65.8 | |||
GAPDH | F | 5' CAT GAG AAG TAT GAC AAC AGC CT 3' | NM_002046 | 113 | 62.0 |
R | 5' AGT CCT TCC ACG ATA CCA AAG T 3' | 62.5 |
The expression level of RPS18 was stable during siRNA treatments. Therefore, RPS18 was designated as the reference gene for relative quantification, through which the expression of endogenous mRNAs from GCT stromal cells was normalized with. Cycle threshold numbers (Ct) were derived from the exponential phase of PCR amplification. Relative changes in mRNA expression were calculated using the comparative
Conditioned media were collected and concentrated using an Amicon Ultra-4 Centrifugal Filter Device (Millipore, Billerica, MA). MMP-13 activity was measured using the SensoLyte Plus 520 MMP-13 Assay Kit (AnaSpec Inc.). Concentrated conditioned media were added into wells of a microtiter plate coated with MMP-13 specific antibodies. After 2-hour incubation at room temperature, APMA was added to each well to activate pro-MMP-13. Then, with the substrate incubation, color development from MMP-13 activity was measured using the plate reader, with an excitation and emission wavelength of 485 ± 20 nm and 530 ± 25 nm, respectively. Concentrated fresh media not exposed to cells were used as a negative control.
GraphPad Prism software (GraphPad Software, Inc., USA) was used for statistical analysis. All data are presented as mean ± standard error of the mean (SEM), and are representative of measurements that were performed on four different GCT patient samples (
To identify which members of the AP-1 family are present in GCT stromal cells, an AP-1 screening assay was used to detect specific transcription factor DNA binding activity in the nuclear extracts. GCT stromal cells stimulated with IL-1
Cell morphology of homogeneous GCT stromal cells induced by IL-1
Expression of AP-1 proteins in the nucleus of GCT stromal cells. The AP-1 screening assay examines the protein level of FosB, c-Fos, Fra-1, Fra-2, JunB, JunD, and c-Jun of the AP-1 family in the nuclear extracts of IL-1
To re-confirm the presence and expression of c-Fos and c-Jun of AP-1 in GCT stromal cells, the baseline mRNA expression level of c-Fos, c-Jun, and Runx2 was determined using real-time PCR. The mRNA expression of these three transcription factors was relatively low into the hundredth level relative to RPS18 expression (Figure
Relative mRNA expression of the Runx2 and AP-1 transcription factors based on real-time RT-PCR. The expression of c-Jun, c-Fos, and Runx2 in GCT stromal cells treated with cytokines IL-1
Next, to further elucidate the role of AP-1 and Runx2 in MMP-13 transcriptional regulation, we depleted various combinations of c-Fos, c-Jun, and Runx2 in the mesenchymal stromal cells of GCT by using RNA interference. Random siRNA served as the negative control. All siRNA treatments (c-Fos, c-Jun, Runx2, and c-Fos + c-Jun, c-Fos + c-Jun + Runx2) were normalized to the random siRNA negative control. The expression of c-Jun was 40–60% suppressed when treated with c-Jun, and c-Fos + c-Jun, c-Fos + c-Jun + Runx2 siRNA, respectively (Figure
The effect of siRNA knockdown on c-Fos, c-Jun, Runx2, MMP-13, and GAPDH mRNA expression in the IL-1
c-Jun
Runx2
MMP-13
GAPDH
To validate the effect of silencing c-Fos, c-Jun, and Runx2 on their downstream target MMP-13 at the translational level, MMP-13 enzyme activity was measured from culture medium collected from the siRNA treatments. The percentage of MMP-13 activity (Figure
The effect of siRNA knockdown on the level of MMP-13 activity in IL-1
The regulation of MMPs plays an important role in tissue remodeling associated with various physiological and pathological processes involving turnover of the extracellular matrix. MMP-13 has a key role in the MMP activation cascade and appears to be critical in bone metabolism, homeostasis, osteoarthritis, and rheumatoid arthritis [
Intervention affecting the regulation of MMPs in pathologic tissues has substantial clinical potential. At least 56 MMP inhibitors have been assessed as candidates in various therapeutic areas, mainly for targeting cancer, arthritis, or cardiovascular diseases [
To get a broader scope of how transcription factors regulate the cytokine-stimulated MMP-13 expression in GCT stromal cells, we specifically examined AP-1 in this study to extend our understanding of Runx2 as a modulator of MMP-13. Both Runx2 and AP-1 have binding sites in the promoter region of MMP-13. Selvamurugan et al. reported that activation of the MMP-13 promoter requires both the AP-1 and Runx2 sites
AP-1 is a transcription factor, which is a heterodimeric protein, composed of proteins belonging to the c-Fos, c-Jun, ATF, and JDP families. It regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, bacterial, and viral infections [
The results of siRNA knockdown in GCT cells showed that silencing c-Jun and Runx2 significantly silenced the expression of MMP-13. However, knocking down all three transcription factors in the c-Fos + c-Jun + Runx2 siRNA condition did not reduce the MMP-13 expression further, indicating a baseline endogenous expression level of MMP-13 controlled by other factors or pathways. This result also suggests that no synergistic interaction happened between the AP-1 and Runx2 transcription factors in MMP-13 regulation. We found the c-Jun element of AP-1 to show higher expression and greater control over MMP-13 expression in GCT stromal cells that the c-Fos element of AP-1. Both c-Fos and c-Jun are end targets of the ERK and JNK pathways such that inhibiting either pathway in our previous study demonstrated a similar trend in subduing MMP-13 expression [
From the results of this study, it is clear that the functional regulation of MMP-13 requires both the AP-1 and Runx2 transcription factors in our GCT stromal cell model. Complex regulatory loops exist in the cells of the skeletal system, which spatially and temporally control the progression of bone and cartilage cell maturation and coordinate it with events in surrounding tissues [
In summary, we have demonstrated that c-Fos and c-Jun of the AP-1 family are expressed by GCT stromal cells. The cytokine-induced MMP-13 expression in these cells is strongly suppressed by various combinations of c-Fos, c-Jun, and Runx2 gene knockdown. Our results indicate that c-Fos, c-Jun, and Runx2 all regulate MMP-13 expression and activity to a certain degree in GCT mesenchymal stromal cells. We propose that cytokines secreted by multinucleated giant cells stimulate MMP-13 production in the GCT stromal cells through both AP-1 and Runx2 transcription factors. The regulation of these transcription factors may therefore serve as targets in treatment strategies for this destructive tumor and other degenerative diseases of the musculoskeletal system where MMP-13 is the most prominently implicated protease. Nevertheless, more evidence is needed to clarify what upstream extracellular or intercellular signals modulate AP-1 and Runx2 in controlling MMP-13 regulation in the human bone environment in order to further our understanding of the physiology of metalloproteinase-induced osteolysis.
The paper received a grant from the Canadian Institutes of Health Research (CIHR) and the Juravinski Cancer Centre Foundation as well as the Hamilton Health Science New Investigator Fund and the McMaster University Surgical Associates Grant.
All authors declare no conflict of interest.