Phosphocitrate (PC) inhibited meniscal calcification and the development of calcium crystal-associated osteoarthritis (OA) in Hartley guinea pigs. However, the mechanisms remain elusive. This study sought to examine the biological activities of PC in the absence of calcium crystals and test the hypothesis that PC is potentially a meniscal protective agent. We found that PC downregulated the expression of many genes classified in cell proliferation, ossification, prostaglandin metabolic process, and wound healing, including bloom syndrome RecQ helicase-like, cell division cycle 7 homolog, cell division cycle 25 homolog C, ankylosis progressive homolog, prostaglandin-endoperoxide synthases-1/cyclooxygenase-1, and plasminogen activator urokinase receptor. In contrast, PC stimulated the expression of many genes classified in fibroblast growth factor receptor signaling pathway, collagen fibril organization, and extracellular structure organization, including fibroblast growth factor 7, collagen type I, alpha 1, and collagen type XI, alpha 1. Consistent with its effect on the expression of genes classified in cell proliferation, collagen fibril organization, and ossification, PC inhibited the proliferation of OA meniscal cells and meniscal cell-mediated calcification while stimulating the production of collagens. These findings indicate that PC is potentially a meniscal-protective agent and a disease-modifying drug for arthritis associated with severe meniscal degeneration.
Osteoarthritis (OA) is one of the most prevalent causes of disability in the aging population and has enormous economic and social consequences. However, existing nonsurgical treatment options only provide symptomatic relief but have no effect on the progression of the underlying disease or cartilage degeneration. The lack of progress in the development of disease-modifying drugs for OA therapy is largely due to our limited understanding of the pathogenesis of OA and our insufficient knowledge about the molecular targets for OA therapy.
Knee OA is not merely an articular cartilage disease, but a disease of the whole joint. An important local factor is the structural integrity of the menisci. In recent years, there has been a dramatic advance in our understanding of the integral role of the menisci for knee function and the consequences of meniscal abnormality on the development of OA. Studies have found that meniscal degeneration is a general feature of knee OA and contributes to joint space narrowing [
Basic calcium phosphate crystal and calcium pyrophosphate dihydrate crystal are the two most common articular calcium crystals. The presence of these crystals in OA articular cartilage and synovial fluid is well recognized. These crystals are also present in knee menisci of patients with end-stage OA [
Phosphocitrate (PC) is a naturally occurring compound originally identified in rat liver mitochondrial extracts and crab hepatopancreas [
Dulbecco’s Modified Eagle Medium, StemPro chondrogenesis differentiation medium, fetal bovine serum, Hank’s balanced salt solution, and stock antibiotic and antimycotic mixture were products of Invitrogen (Carlsbad, CA, USA). PC was synthesized according to the procedure described [
OA meniscal cells were prepared from menisci derived from patients with end-stage OA. Briefly, the medial menisci derived from patients with end-stage OA were processed to remove fatty and synovial tissues, minced into small pieces, and cultured in 100 mm plates at 37°C in medium containing 0.5% antibiotic/antimycotic solution and 10% serum. Every three or four days, the culture medium was changed. When the cells reached 70% confluence, they were passaged and maintained in medium containing 10% serum. Human foreskin fibroblasts were obtained from American Type Culture Collection (CRL-2429, Manassas, VA, USA). OA meniscal cells prepared from three OA patients were used in this study. OA menisci were collected with the approval of the authors’ Institutional Review Board from OA patients undergoing knee joint replacement surgery. The need for informed consent was waived because those menisci were surgical waste, and no private patient information was collected.
OA meniscal cells derived from three OA patients were harvested from cell culture plates and mixed and replated in four 100 mm cell culture plates at 90% confluence. On the second day, medium containing 1% serum was added. Twenty-four hours later, the medium in two plates was replaced with medium containing 1% serum and PC (1 mM), and the medium in the other two plates was replaced with medium containing 1% serum without PC. Twenty-four hours later, total RNA was extracted from these cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using Oligotex kit (Qiagen, Valencia, CA, USA). We repeated the experiment twice. Microarray was performed using these RNA samples (total six RNA samples).
RNA samples extracted from three independent experiments were used for microarray analysis experiments. Briefly, double-stranded DNA was synthesized using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, San Diego, CA, USA). The DNA product was purified using GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA, USA). cRNA was synthesized and biotin-labeled using BioArray high yield RNA transcript labeling kit (Enzo Life Sciences, Farmingdale, NY, USA). The cRNA product was purified using GeneChip Sample Cleanup Module and subsequently chemically fragmented. The fragmented and biotinylated cRNA was hybridized to HG-U133_Plus_2 GeneChip using Affymetrix Fluidics Station 400 (Affymetrix, Santa Clara, CA, USA). The fluorescent signals were quantified during two scans by Agilent Gene Array Scanner G2500A (Agilent Technologies, Palo Alto, CA, USA) and GeneChip operating Software (Affymetrix, Santa Clara, CA, USA). Genesifter (VizX Labs, Seattle, WA, USA) was used for the analysis of differential gene expression and gene ontology.
After microarray analyses, we mixed the RNA samples extracted from PC-treated OA meniscal cells (PC-treated RNA sample) and the RNA samples extracted from untreated OA meniscal cells (untreated RNA sample) and performed RT-PCR experiments. Briefly, cDNA was synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems, University Park, IL, USA) using the RNA samples described. Quantification of relative transcript levels for selected genes and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed using ABI7000 Real-Time PCR system (Applied Biosystems, University Park, IL, USA). TaqMan Gene Expression assay (Applied Biosystems, University Park, IL, USA) was used. CDNA samples were amplified with an initial Taq DNA polymerase activation step at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing at 60°C for one minute. Fold change was calculated, and the expression level of the genes to be examined was normalized to the expression level of GAPDH. RT-PCR experiment was performed in triplicates using the same RNA sample.
OA meniscal cells (2 × 104) were plated in six well cluster plates and cultured in medium containing 10% serum in the presence of increasing amounts of PC (triplicates). Medium was changed every three days until the OA meniscal cells in the wells without PC reached 85% confluence. Cells were then harvested and cell numbers were determined using a hemocytometer. This experiment was repeated 3 times using OA meniscal cells derived from different patients. This proliferation assay was also performed using human primary foreskin fibroblasts.
OA meniscal cells were harvested from several 100 mm culture plates and suspended in medium containing 10% serum. For preparing a micromass, a droplet of the cell suspension containing 6 × 106 cells was placed in a well of a 24-well plate. After placing all droplets, the plate was incubated for 4 hours at 37°C in a tissue culture incubator. These micromasses were then fed with StemPro chondrogenesis differentiation medium with PC (1 mM) or without PC every three days throughout the experiment for 14 days. Each well was then rinsed twice with 500
OA meniscal cells were plated in twenty-four well plates at 90% confluence. The next day, medium was replaced with StemPro chondrogenesis differentiation medium containing 1 mM adenosine-5′-triphosphate (ATP) in the absence or presence of PC (1 mM). The cells were cultured for 14 days and fed with StemPro chondrogenesis differentiation medium containing ATP every three or four days throughout the experiment. At the end of the experimental period, media were removed. Calcification was examined using alizarin red.
For cell proliferation, data are expressed as the mean ± SD, and the difference between two groups was analyzed using Student’s
We performed three microarray experiments (PC-treated RNA sample I and untreated RNA sample I; PC-treated RNA sample II and untreated RNA sample II; PC-treated RNA sample III and untreated RNA sample III) as described. The results of the three microarray experiments were quite similar. The results of the first microarray experiment showed that of the more than 50,000 transcripts, 2445 transcripts displayed significant differential expression (more than 1.6-fold changes) in the PC-treated OA meniscal cells compared with the untreated OA meniscal cells. A total of 1795 transcripts displayed decreased expression and 650 transcripts displayed increased expression. The genes that fell into specific biological processes previously implicated in OA, or suspected to have a role in OA are listed in Tables
Differentially expressed genes in PC-treated via untreated OA meniscal cells.
Biological |
Gene |
Gene ID | Differential |
Description |
---|---|---|---|---|
Cell proliferation | BLM | NM_000057 | −7.41 | Bloom syndrome, RecQ helicase-like |
NDP | NM_000266 | −4.68 | Norrie disease (pseudoglioma) | |
HELLS | AF155827 | −4.46 | Helicase, lymphoid specific | |
E2F7 | AI341146 | −4.12 | E2F transcription factor 7 | |
CDC7 | NM_003503 | −4.11 | Cell division cycle 7 homolog ( | |
CDCA7 | AY029179 | −2.71 | Cell division cycle-associated 7 | |
CDC25C | NM_001790 | −2.36 | Cell division cycle 25 homolog C ( | |
BRCA1 | AF005068 | −3.60 | Breast cancer 1, early onset | |
BRCA2 | X95152 | −3.30 | Breast cancer 2, early onset | |
PRKRA | AA279462 | −3.34 | PKinase, interferon-indu double-stranded RNA-dependent activator | |
HHIP | AK098525 | −3.28 | Hedgehog interacting protein | |
CHEK1 | AA224205 | −3.11 | CHK1 checkpoint homolog ( | |
PTPRK | AU145587 | −3.08 | Protein tyrosine phosphatase, receptor type, |
|
GINS1 | NM_021067 | −3.07 | GINS complex subunit 1 (Psf1 homolog) | |
TCF19 | BC002493 | −2.93 | Transcription factor 19 | |
MKI67 | AU147044 | −2.92 | Antigen identified by monoclonal antibody Ki-67 | |
PDS5B | AK026889 | −2.88 | PDS5, regulator of cohesion maintenance, homolog B | |
UHRF1 | AK025578 | −2.77 | Ubiquitin-like with PHD and ring finger domains 1 | |
AURKB | AB011446 | −2.64 | Aurora kinase B | |
MKI67 | AU132185 | −2.64 | Antigen identified by monoclonal antibody Ki-67 | |
FIGNL1 | NM_022116 | −2.62 | Fidgetin-like 1 | |
KIF15 | NM_020242 | −2.60 | Kinesin family member 15 | |
LRP6 | NM_002336 | −2.55 | Low-density lipoprotein receptor-related protein 6 | |
ANXA1 | AU155094 | −2.47 | Annexin A1 | |
DDX11 | U33833 | −2.47 | DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 | |
FANCA | AW083279 | −2.46 | Fanconi anemia, complementation group A | |
SPRY2 | NM_005842 | −2.43 | Sprouty homolog 2 ( | |
RECQL4 | NM_004260 | −2.43 | RecQ protein-like 4 | |
NTN1 | BF591483 | −2.42 | Netrin 1 | |
ADRB2 | NM_000024 | −2.41 | Adrenergic, beta-2, receptor, surface | |
CUL4A | AU155661 | −2.40 | Cullin 4A | |
DLC1 | NM_024767 | −2.39 | Deleted in liver cancer 1 | |
STIL | NM_003035 | −2.39 | SCL/TAL1 interrupting locus | |
CHEK1 | NM_001274 | −2.37 | CHK1 checkpoint homolog ( | |
TIMELESS | NM_003920 | −2.37 | Timeless homolog ( | |
SMAD4 | AL832789 | −2.23 | SMAD family member 4 | |
SMAD1 | NM_015583 | −2.19 | SMAD family member 1 | |
PCNA | NM_002592 | −2.18 | Proliferating cell nuclear antigen | |
PRKCD | NM_006254 | −2.17 | Protein kinase C, delta | |
RBBP4 | AI972451 | −2.16 | Retinoblastoma binding protein 4 | |
PTGS1 | NM_000962 | −2.13 | Prostaglandin-endoperoxide synthase 1 | |
ASPM | NM_018123 | −2.13 | Abnormal spindle (asp) homolog, microcephaly associated | |
GLMN | AA814383 | −2.11 | Glomulin, FKBP associated protein | |
NASP | NM_002482 | −2.11 | Nuclear autoantigenic sperm protein (histone binding) | |
CCNA2 | NM_001237 | −2.09 | Cyclin A2 | |
TBX2 | AW173045 | −2.08 | T-box 2 | |
KIF2C | U63743 | −2.08 | Kinesin family member 2C | |
PDCD1LG2 | AF329193 | −2.06 | Programmed cell death 1 ligand 2 | |
BUB1B | NM_001211 | −2.05 | Budding uninhibited by benzimidazoles 1 homolog beta (yeast) | |
POLA1 | NM_016937 | −2.04 | Polymerase (DNA directed), alpha 1, catalytic subunit | |
TACC3 | NM_006342 | −2.02 | Transforming, acidic coiled-coil containing protein 3 | |
CDK2 | M68520 | −2.01 | Cyclin-dependent kinase 2 | |
DLX5 | NM_005221 | 2.99 | Distal-less homeobox 5 | |
VCAM1 | NM_001078 | 2.88 | Vascular cell adhesion molecule 1 | |
ADORA1 | NM_000674 | 2.48 | Adenosine A1 receptor | |
TNFRSF9 | NM_001561 | 2.38 | Tumor necrosis factor receptor superfamily, member 9 | |
FGF9 | NM_002010 | 2.38 | Fibroblast growth factor 9 (glia-activating factor) | |
FABP3 | NM_004102 | 2.24 | Fatty acid binding protein 3, muscle, and heart | |
FGFR2 | M87771 | 2.20 | Fibroblast growth factor receptor 2 | |
BAMBI | NM_012342 | 2.13 | BMP and activin membrane-bound inhibitor homolog | |
CD24 | L33930 | 2.09 | CD24 molecule | |
HSF1 | AI393937 | 2.07 | Heat shock transcription factor 1 | |
| ||||
Ossification | COL13A1 | M33653 | −3.39 | Collagen, type XIII, alpha 1 |
SATB2 | AK025127 | −3.13 | SATB homeobox 2 | |
ADRB2 | NM_000024 | −2.41 | Adrenergic, beta-2, receptor, surface | |
SMAD1 | NM_015583 | −2.19 | SMAD family member 1 | |
ENPP1 | BF591996 | −2.02 | Ectonucleotide pyrophosphatase/phosphodiesterase 1 | |
BMPR1B | AA935461 | −1.93 | Bone morphogenetic protein receptor, type IB | |
NAB1 | AF045452 | −1.87 | NGFI-A-binding protein 1 (EGR1-binding protein 1) | |
GNAS | AA810695 | −1.86 | GNAS complex locus | |
FGF18 | BC006245 | −1.73 | Fibroblast growth factor 18 | |
TNFRSF11A | AW026379 | −1.67 | Tumor necrosis factor receptor superfamily, member 11a | |
EGFR | K03193 | −1.61 | Epidermal growth factor receptor | |
PLA2G4A | M68874 | −1.61 | Phospholipase A2, group IVA (cytosolic, calcium dependent) | |
ANKH | T99215 | −1.60 | Ankylosis, progressive homolog (mouse) | |
DLX5 | NM_005221 | 2.99 | Distal-less homeobox 5 | |
FGF9 | NM_002010 | 2.38 | Fibroblast growth factor 9 (glia-activating factor) | |
IGFBP5 | R73554 | 1.78 | Insulin-like growth factor-binding protein 5 | |
GABBR1 | N45228 | 1.72 | Gamma-aminobutyric acid (GABA) B receptor, 1 | |
MMP14 | NM_004995 | 1.72 | Matrix metallopeptidase 14 (membrane inserted) | |
TUFT1 | NM_020127 | 1.66 | Tuftelin 1 | |
COL1A1 | AI743621 | 1.62 | Collagen, type I, alpha 1 | |
MGP | NM_000900 | 1.61 | Matrix Gla protein | |
SMAD3 | BF971416 | 1.60 | SMAD family member 3 | |
| ||||
BMP signaling pathway | UBE2D3 | AI239832 | −2.44 | Ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homolog, yeast) |
ZFYVE16 | BC032227 | −2.43 | Zinc finger, FYVE domain containing 16 | |
SMAD4 | AL832789 | −2.23 | SMAD family member 4 | |
SMAD1 | NM_015583 | −2.19 | SMAD family member 1 | |
BMPR1B | AA935461 | −1.93 | Bone morphogenetic protein receptor, type IB | |
HIPK2 | AW300045 | −1.91 | Homeodomain interacting protein kinase 2 | |
MSX1 | AI421295 | −1.84 | Msh homeobox 1 | |
GREM2 | NM_022469 | −1.73 | Gremlin 2, cysteine knot superfamily, homolog (Xenopus laevis) | |
| ||||
Prostaglandin metabolic process | PTGS1 | NM_000962 | −2.13 | Prostaglandin-endoperoxide synthase 1 |
AKR1C2 | BF508244 | −1.91 | Aldo-keto reductase family 1, member C2 | |
PLA2G4A | M68874 | −1.61 | Phospholipase A2, group IVA (cytosolic, calcium dependent) | |
PTGS2 | NM_000963 | − |
Prostaglandin-endoperoxide synthase 2 | |
PTGIS | D38145 | 1.79 | Prostaglandin I2 (prostacyclin) synthase | |
PDPN | AW590196 | 1.61 | Podoplanin | |
| ||||
Response to wounding | THBD | AW119113 | −4.24 | Thrombomodulin |
PLAT | NM_000930 | −3.12 | Plasminogen activator, tissue | |
TFPI | J03225 | −2.66 | Tissue factor pathway inhibitor | |
TFPI2 | AL574096 | −2.38 | Tissue factor pathway inhibitor 2 | |
SDC1 | NM_002997 | −2.38 | Syndecan 1 | |
SMAD1 | NM_015583 | −2.19 | SMAD family member 1 | |
PLAUR | AY029180 | −1.94 | Plasminogen activator, urokinase receptor | |
TMPRSS6 | AI912086 | 1.96 | Transmembrane protease, serine 6 | |
JUB | NM_032876 | 1.92 | Jub, ajuba homolog (Xenopus laevis) |
Differentially expressed genes in PC-treated via untreated OA meniscal cells.
Biological |
Gene |
Gene ID | Differential |
Description |
---|---|---|---|---|
FGF receptor signaling pathway | FGF9 | NM_002010 | 2.38 | Fibroblast growth factor 9 (glia-activating factor) |
FGFR2 | M87771 | 2.20 | Fibroblast growth factor receptor 2 | |
FGF7 | NM_002009 | 1.83 | Fibroblast growth factor 7 (keratinocyte growth factor) | |
NDST1 | AL526632 | 1.71 | N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1 | |
THBS1 | AI812030 | 1.68 | Thrombospondin 1 | |
HHIP | AK098525 | −3.28 | Hedgehog interacting protein | |
FGF18 | BC006245 | −1.73 | Fibroblast growth factor 18 | |
| ||||
Collagen fibril organization | COL14A1 | M64108 | 2.06 | Collagen, type XIV, alpha 1 |
COL11A1 | J04177 | 1.82 | Collagen, type XI, alpha 1 | |
COL1A1 | AI743621 | 1.62 | Collagen, type I, alpha 1 | |
SERPINH1 | NM_004353 | 1.66 | Serpin peptidase inhibitor, clade H, member 1 | |
DPT | AI146848 | 1.65 | Dermatopontin | |
TRAM2 | BC028121 | 1.60 | Translocation-associated membrane protein 2 | |
COL5A1 | AI983428 |
|
Collagen, type V, alpha 1 | |
COL5A2 | NM_000393 |
|
Collagen, type V, alpha 2 | |
| ||||
Extracellular structure organization | CACNA1A | NM_023035 | 2.55 | Calcium channel, voltage dependent, P/Q type, alpha 1A subunit |
COL14A1 | M64108 | 2.06 | Collagen, type XIV, alpha 1 | |
ECM2 | NM_001393 | 2.04 | Extracellular matrix protein 2, female organ, and adipocyte specific | |
MYO6 | AA877789 | 1.98 | Myosin VI | |
TMPRSS6 | AI912086 | 1.96 | Transmembrane protease, serine 6 | |
COL11A1 | J04177 | 1.82 | Collagen, type XI, alpha 1 | |
CRISPLD2 | AL136861 | 1.74 | Cysteine-rich secretory protein LCCL domain containing 2 | |
HSD17B12 | BC012536 | 1.68 | Hydroxysteroid (17-beta) dehydrogenase 12 | |
SERPINH1 | NM_004353 | 1.66 | Serpin peptidase inhibitor, clade H, member 1 | |
DCN | AI336924 | 1.65 | Decorin | |
DPT | AI146848 | 1.65 | Dermatopontin | |
COL1A1 | AI743621 | 1.62 | Collagen, type I, alpha 1 | |
APLP1 | U48437 | 1.61 | Amyloid beta (A4) precursor-like protein 1 | |
NFASC | AI821777 | −1.99 | Neurofascin homolog (chicken) | |
MMP9 | NM_004994 | −1.91 | Matrix metallopeptidase 9 | |
| ||||
Inflammatory response | ADORA1 | NM_000674 | 2.48 | Adenosine A1 receptor |
CD24 | L33930 | 2.09 | CD24 molecule | |
BCL6 | S67779 | 1.98 | B-cell CLL/lymphoma 6 | |
NGF | NM_002506 | 1.84 | Nerve growth factor (beta polypeptide) | |
CFD | NM_001928 | 1.82 | Complement factor D (adipsin) | |
ITGB2 | NM_000211 | 1.80 | Integrin, beta 2 | |
CFI | BC020718 | 1.74 | Complement factor I | |
NDST1 | AL526632 | 1.71 | N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1 | |
IL17C | AF152099 | 1.70 | Interleukin 17C | |
THBS1 | AI812030 | 1.68 | Thrombospondin 1 | |
SERPINA3 | NM_001085 | 1.67 | Serpin peptidase inhibitor, clade A, member 3 | |
AGTR1 | NM_004835 | 1.65 | Angiotensin II receptor, type 1 | |
TFRC | N76327 | 1.64 | Transferrin receptor (p90, CD71) | |
PDPN | AW590196 | 1.61 | Podoplanin | |
MASP1 | AI274095 | 1.61 | Mannan-binding lectin serine peptidase 1 | |
NFKBIZ | BE646573 | −2.58 | NFKb inhibitor, zeta | |
ANXA1 | AU155094 | −2.47 | Annexin A1 | |
ADRB2 | NM_000024 | −2.41 | Adrenergic, beta-2, receptor, surface | |
SMAD1 | NM_015583 | −2.19 | SMAD family member 1 | |
CXCL6 | NM_002993 | −2.19 | Chemokine (C-X-C motif) ligand 6 | |
C2 | NM_000063 | −2.18 | Complement component 2 | |
BMPR1B | AA935461 | −1.93 | Bone morphogenetic protein receptor, type IB | |
TNFRSF1B | NM_001066 | −1.74 | Tumor necrosis factor receptor superfamily, member 1B | |
EDNRA | NM_001957 | −1.67 | Endothelin receptor type A | |
PLA2G4A | M68874 | −1.61 | Phospholipase A2, group IVA (cytosolic, calcium dependent) | |
GPR68 | AI805006 | −1.60 | G protein-coupled receptor 68 |
As shown in Table
The expressions of many genes classified in ossification and bone morphogenetic protein (BMP) signaling pathway were also downregulated by PC (Table
In addition, the expression of several genes classified in prostaglandin metabolic process and respond to wounding was downregulated by PC (Table
Many genes that were upregulated by PC fell into the biological processes of fibroblast growth factor (FGF) receptor signaling pathway, collagen fibril organization, and extracellular structure organization (Table
Finally, of the 26 genes classified in inflammatory response, the expression of 15 genes, including adenosine A1 receptor (ADORA1; 2.48-fold change), CD24 molecule (CD24; 2.09-fold change), and B-cell CLL/lymphoma 6 (BCL6; 1.98-fold change), was upregulated by PC. The expression of 11 genes, including NF-kappaB inhibitor zeta (NFKBIZ; −2.58-fold change), adrenergic, beta-2, receptor (ADRB2; −2.41-fold change), and chemokine (C-X-C motif) ligand 6 (CXCL6; −2.19-fold change), was downregulated by PC (Table
Real-time RT-PCR was used to confirm expression of selected genes. The results are listed in Table
Differential expression confirmed by real-time RT-PCR.
Gene name | Gene ID | Differential |
Differential |
Differential |
---|---|---|---|---|
BLM | NM_000057 | −7.41 | −5.54 | −6.54 |
HELLS | AF155827 | −4.46 | −4.17 | −4.87 |
CDC25C | NM_001790 | −2.36 | −2.18 | −2.92 |
CDC6 | NM_001254 | −2.00 | −2.20 | −3.41 |
CCNE2 | NM_004702 | −3.51 | −2.94 | −3.46 |
CCNA2 | A1346350 | −2.00 | −2.34 | −1.78 |
ANKH | T99215 | −1.60 | −1.42 | −1.86 |
PTGS1 | NM_000962 | −2.13 | −2.43 | −2.98 |
THBD | AW119113 | −4.24 | −4.51 | −3.79 |
FGF7 | NM_002009 | 1.83 | 1.96 | 2.32 |
FGF9 | NM_002010 | 2.38 | 2.47 | 2.70 |
COL11A1 | J04177 | 1.82 | 1.96 | 2.01 |
ECM2 | NM_001393 | 2.04 | 2.71 | 2.15 |
IGFBP5 | R73554 | 1.78 | 1.95 | 2.34 |
CD24 | L33930 | 2.09 | 2.32 | 3.10 |
The specific downregulatory effect of PC on the expression of genes classified in cell proliferation suggests that PC may inhibit the proliferation of OA meniscal cells. To test this, we cultured OA meniscal cells in the absence and presence of PC for 9 days and then determined the cell number using a hemocytometer. Indeed, we found that PC inhibited the proliferation of OA meniscal cells (Figure
PC inhibited the proliferation of OA meniscal cells. OA meniscal cells and human foreskin fibroblast were plated in six-well cluster plates and cultured in the presence or absence of PC. (a) There were about 55% fewer OA meniscal cells in the PC-treated (1 mM PC) wells than that in the untreated wells (the right bar group;
The upregulatory effect of PC on the expression of genes classified in collagen fibril organization, including COL1A1 (1.62-fold change), COL11A1 (1.82-fold change), COL14A1 (2.06-fold change), COL5A1 (1.52-fold change), and COL5A2 (1.44-fold change), suggests that PC may stimulate the production of collagens by the OA meniscal cells. To examine this, we prepared micromasses of OA meniscal cells (triplicates) and examined the production of collagens using picrosirius red staining. The results confirmed that PC stimulated the production of collagens by OA meniscal cells. Representative images of picrosirius red staining are shown in Figure
PC stimulated the production of collagens. Micromasses of OA meniscal cells were cultured in the absence of PC ((a); magnification 10x) or the presence of 1 mM PC ((b); magnification 10x) for 14 days. These micromasses were then processed and stained with picrosirius red. Note the much stronger picrosirius red staining in the PC-treated micromass than that in the untreated micromass.
These micromasses were also stained with alizarin red for calcium deposits. Representative images of alizarin red staining are shown in Figure
PC inhibited OA meniscal cells-mediated calcification in micromass culture. Micromasses of OA meniscal cells were stained with alizarin red. In the absence of PC, calcium deposits were detected ((a); magnification 10x). In the presence of 1 mM PC, calcium deposits were abolished ((b); magnification 10x).
The inhibitory effect of PC on OA meniscal cell-mediated calcification was also examined using monolayer culture. As shown in Figure
PC inhibited OA meniscal cells-mediated calcification in monolayer culture. In the absence of PC, calcium deposits were formed ((a); magnification 20x). In the presence of 1 mM PC, no calcium deposits were formed ((b); magnification 20x).
Increased number and size of cell clusters are the hallmark histological feature of OA articular cartilage [
Studies consistently reported apoptotic cells in cell clusters [
Several previous observations provide support for this potential mechanism. For example, studies found that COL13 was increased in OA articular cartilage and that transgenic mice overexpressing COL13 had abnormally high bone mineral density [
Early studies demonstrated that MGP was a calcification inhibitor and that inhibition of IGFBP5 breakdown reduced articular cartilage loss in an experimental OA [
FGF signaling pathway plays an important role in the regulation of osteogenesis and chondrogenesis. Studies found that FGF18 induced chondrocyte hypertrophy and mineralization [
Severe loss of collagen occurs in OA menisci [
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat arthritis. Traditional NSAIDs inhibit both PTGS1/Cox-1 and PTGS2/Cox-2, while new generation of NSAIDs selectively inhibits PTGS2/Cox-2. In this study, we demonstrated that PC downregulated the expression of PTGS1/Cox-1 (Table
Interestingly, many genes classified in the inflammatory response were upregulated by PC, including adenosine A1 receptor ADORA1 (2.48-fold change), CD24 (2.09-fold change), and BCL6 (1.98-fold change). It is worth noting that previous studies demonstrated that the activation of adenosine receptors reduced inflammation and joint destruction in a rat adjuvant-induced arthritis, CD24 repressed tissue damage-induced immune response, and that BCL6 inhibited the expression of chemokines and attenuated allergic airway inflammation [
The genes downregulated by PC included NFKBIZ (−2.58-fold change), ADRB2 (−2.41-fold change), and CXCL6 (−2.19-fold change). Interestingly, studies demonstrated that NFKBIZ mediated IL-6 production, ADRB2 antagonists reduced the severity of arthritis, and that anti-CXCL16 antibody inhibited infiltration of inflammatory cells and arthritis [
These specific biological activities of PC are intrinsic properties of PC and are not dependent on the presence of calcium crystals. PC is potentially an anti-inflammation and meniscal protective agent. PC is not only potentially a disease-modifying drug for crystal-associated OA but also potentially a disease-modifying drug for arthritis associated with severe meniscal degeneration. The findings presented in this study provide further support for the development of PC, and/or its analogues, as disease-modifying drugs for OA therapy.
The authors declare no conflict of interests.
This study is supported in part by an NC Biotech Center Grant, a Charlotte-Mecklenburg Education and Research Foundation Grant, and a Mecklenburg County Medical Society Smith Arthritis Fund Grant (to Yubo Sun). This study was performed at Carolinas Medical Center, Charlotte, NC, USA. The authors would like to thank Natalia Zinchenko for her help with histological examinations of micromasses.