GPNMB expression is increased in high-grade OA cartilage and recombinant GPNMB reduces catabolic gene expression in human HTB-94 cell culture and cartilage damage in human cartilage explants.

Human articular cartilage was isolated from low-grade and high-grade OA joints and mRNA expression of (A) ACAN and (B) GPNMB were determined via RT-qPCR. HTB-94 cells treated with combination of recombinant GPNMB and IL-1β demonstrated significantly decreased expression of (C) Mmp-9 (D) Mmp-13 and (E) IL-6 mRNAs. Combined recombinant GPNMB and IL-1β treatment significantly reduced cartilage degradation in human explant cultures compared to IL-1β treatment alone in Safranin-O-stained human cartilage explants (F). Scale bar= 200µm in top panels and 100µm in magnified lower images. Data presented as Mean ± SEM (n=7). **= p<0.01, ***=p<0.001, ****=p<0.0001.

Gpnmb expression is induced with IL-1β proinflammatory stimulation, reduced in DBA/2J mice and recombinant GPNMB inhibits catabolic expression in DBA/2J GPNMB+ and DBA/2J chondrocytes.

(A) Gpnmb expression is increased with pro-inflammatory IL-1β treatment. Gpnmb expression (B) and protein (C, D) is significantly reduced in DBA/2J mice. Recombinant GPNMB treatment does not affect chondrocyte proliferation (E) or viability (F). Recombinant GPNMB inhibits Mmp-3, Mmp-9, Mmp-13 expression in DBA/2J GPNMB+ (G-I) and DBA/2J (J-L) chondrocytes and reduces Adamts-4 (M,N) and IL-6 (O,P) expression. Scale bar= 200µm. Data presented as Mean ± SEM (n=6). *=p<0.05, **= p<0.01, ***=p<0.001, ****=p<0.0001.

Recombinant GPNMB treatment does not alter anabolic gene expression.

Anabolic gene expression was measured in DBA/2J GPNMB+ and DBA/2J chondrocytes by RT-qPCR. Sox-9 mRNA was decreased with IL-1β treatment in DBA/2J GPNMB+ (A) and DBA/2J (D) chondrocytes and was not rescued following treatment with recombinant GPNMB. Col2α1 expression was decreased with IL-1β treatment and was not increased significantly upon addition of recombinant GPNMB in DBA/2J GPNMB+ (B) or DBA/2J (E) chondrocytes. Acan mRNA expression decreased with IL-1β treatment and was not rescued with the addition of recombinant GPNMB in DBA/2J GPNMB+ (C) or DBA/2J (F) chondrocytes. Data presented as Mean ± SEM (n=6). ns= not significant, **= p<0.01, ***=p<0.001, ****=p<0.0001.

DMM surgery resulted in cartilage fibrillation after 12 weeks in C57BL6/J mice and damage was accelerated in DBA/2J mice following DMM.

DMM surgery was validated in C57BL/6J mice. Cartilage was intact in sham operated joints (A) while DMM joints displayed superficial and intermediate cartilage damage (B) (tibia shown). (C) OARSI scores for sham and DMM operated joints. DBA/2J GPNMB+ (D) and DBA/2J (E) mice, which lack functional GPNMB protein, were subjected to sham and DMM surgery and joints were stained with Toluidine blue. Black arrows indicate damaged areas. (F) OARSI scores for femur (MFC) and tibia (MTP) cartilage damage of DBA/2J GPNMB+ and DBA/2J mice. Scale bar= 200µm in C57BL/6J joints and top panels and 100µm in magnified lower images (D, E). Images shown are representative and OARSI scorings from multiple images of n=7 each C57BL/6J, DBA/2J GPNMB+ and DBA/2J mice. Data presented as Mean ± SEM (n=6). *p<0.05, **= p<0.01.

ACAN expression is severely depleted, and IL-6 expression elevated following DMM in DBA/2J mice.

ACAN expression was not significantly altered in sham surgery mice (A-C) but was reduced following DMM in DBA/2J GPNMB+ (A, D) and was severely depleted in DBA/2J (B, D) cartilage. Endogenous IL-6 expression is increased in DBA/2J animals without DMM (E-G). DBA/2J GPNMB+ (E, H) and DBA/2J (B, H) cartilage showed an increase in IL-6 following DMM surgery. Scale bar= 200µm top panel and 100µm in magnified lower images. Images shown are representative and OARSI scores are taken multiple images of n=5-7 each DBA/2J GPNMB+ and DBA/2J mice. Data presented as Mean ± SEM. *p<0.05, **p<0.01.

Recombinant GPNMB inhibits the MAPK signaling pathway in primary chondrocytes.

Immunoblotting of primary chondrocytes following IL-1β stimulation with and without recombinant GPNMB show GPNMB inhibits pERK at (A) 30 and (B) 50 minutes post-treatment. Densitometric analyses of pERK were done at (C) 30 and (D) 50 minutes. Experiments were repeated three times with similar results. Data presented as Mean ±SEM. ****=p<0.0001, ns= not significant.

Schematic of the proposed mechanism of GPNMB anti-inflammatory function in chondrocytes.

(A) In healthy chondrocytes, GPNMB functions without inflammatory stimuli to modulate catabolic chemokines and cytokines and maintain cartilage homeostasis. (B) In the presence of inflammatory stimuli (IL-1β), GPNMB acts to modulate MAPK to reduce expression of catabolic chemokines and cytokines and reduce cartilage damage. (C) In DBA/2J mice with non-functional GPNMB, there is no inhibition of inflammatory molecules leading to increased catabolic expression and increased cartilage damage.

qPCR primers

Table of Primary Antibodies

The structure of full length, recombinant and mutant GPNMB proteins.

The full length GPNMB is 574 amino acids and has several domains including a N-terminal extracellular domain signal peptide (SS, 1-22), an arginine-glycine-aspartic acid (RGD), a polycystic kidney disease (PKD), and a proline rich repeat (PRRD) domain. The transmembrane domain (TM) of GPNMB is 20 amino acids (503-523). The C-terminal intracellular domain holds an ITAM motif and Di-leucine signal. The recombinant form of GPNMB represents the N-terminal portion of the molecule (23-502) and is derived from a mouse myeloma cell line. The DBA/2J mutant GPNMB is a truncated version of the protein (1-150).

Endogenous levels of catabolic genes are not significantly different between DBA/2J GPNMB+ and DBA/2J primary chondrocytes.

RT-qPCR analyses of (A) Mmp-3, (B) Mmp-9, (C) Mmp-13, (D) Adamts-4 and (E) Il-6 expression detected no significant differences in endogenous mRNA expression prior to treatment. Experiments were repeated three times in triplicate with similar results. Data presented as Mean ± SEM; ns = not significant.

Endogenous levels of anabolic genes are not significantly different between DBA/2J GPNMB+ (control) and DBA/2J primary chondrocytes.

RT-qPCR analyses of endogenous anabolic gene expression found no significant differences in (A) Sox-9, (B) Type-II collagen (Col2α1) or (C) ACAN expression. Experiments were repeated three times in triplicate with similar results. Data presented as Mean ± SEM; ns = not significant.