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, 225 (1), 152-67

Role of TNF Alpha and PLF in Bone Remodeling in a Rat Model of Repetitive Reaching and Grasping

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Role of TNF Alpha and PLF in Bone Remodeling in a Rat Model of Repetitive Reaching and Grasping

Shobha Rani et al. J Cell Physiol.

Abstract

We have previously developed a voluntary rat model of highly repetitive reaching that provides an opportunity to study effects of non-weight bearing muscular loads on bone and mechanisms of naturally occurring inflammation on upper limb tissues in vivo. In this study, we investigated the relationship between inflammatory cytokines and matricellular proteins (Periostin-like-factor, PLF, and connective tissue growth factor, CTGF) using our model. We also examined the relationship between inflammatory cytokines, PLF and bone formation processes. Rats underwent initial training for 5 weeks, and then performed a high repetition high force (HRHF) task (12 reaches/min, 60% maximum grip force, 2 h/day, 3 days/week) for 6 weeks. We then examined the effect of training or task performance with or without treatment with a rat specific TNFalpha antibody on inflammatory cytokines, osteocalcin (a bone formation marker), PLF, CTGF, and behavioral indicators of pain or discomfort. The HRHF task decreased grip strength and induced forepaw mechanical hypersensitivity in both trained control and 6-week HRHF animals. Two weeks of anti-TNFalpha treatment improved grip strength in both groups, but did not ameliorate forepaw hypersensitivity. Moreover, anti-TNFalpha treatment attenuated task-induced increases in inflammatory cytokines (TNFalpha, IL-1alpha, and MIP2 in serum; TNFalpha in forelimb bone and muscles) and serum osteocalcin in 6-week HRHF animals. PLF levels in forelimb bones and flexor digitorum muscles increased significantly in 6-week HRHF animals, increases attenuated by anti-TNFalpha treatment. CTGF levels were unaffected by task performance or anti-TNFalpha treatment in 6-week HRHF muscles. In primary osteoblast cultures, TNFalpha, MIP2 and MIP3a treatment increased PLF levels in a dose dependent manner. Also in primary osteoblast cultures, increased PLF promoted proliferation and differentiation, the latter assessed by measuring Runx2, alkaline phosphatase (ALP) and osteocalcin mRNA levels; ALP activity; as well as calcium deposition and mineralization. Increased PLF also promoted cell adhesion in MC3T3-E1 osteoblast-like cell cultures. Thus, tissue loading in vivo resulted in increased TNFalpha, which increased PLF, which then induced anabolic bone formation, the latter results confirmed in vitro.

Figures

Figure 1
Figure 1. Anti-TNF intervention for 2 weeks attenuated increased cytokines/chemokines and osteocalcin levels in high repetition high force (HRHF) task animals
Bar graphs showing A,B) tumor necrosis factor alpha (TNFα) and interleukin 1 alpha (IL-1α) levels in serum, C,D) chemokines macrophage inflammatory protein 2 and 3a (MIP2 and MIP3a) levels in serum, E) anti-inflammatory cytokine, IL-10, in serum, F) osteocalcin, a bone formation marker, levels in serum, F) TNFα levels in forelimb flexor digitorum muscles, and G) TNFα levels in forelimb radius and ulna bones,. Data shown for normal controls (NC), trained controls with and without anti-TNF treatment (TC/aTNF and TC, respectively), and 6 week HRHF animals with or without anti-TNF treatment (6HRHF/aTNF and 6HRHF, respectively), in the last 2 weeks before euthanasia. ap<0.01 compared to NC; bp<0.05 compared to NC; cp<0.05 compared to TC; dp<0.01 compared to 6HRHF.
Figure 2
Figure 2. Anti-TNF intervention for 2 weeks improved grip strength significantly but not ameliorate forepaw mechanical hypersensitivity in high repetition high force (HRHF) task animals
A) Forelimb grip strength and B) cutaneous mechanical sensitivity using Von Frey test (withdrawal threshold) is shown for normal controls (NC), trained controls (TC) with or without anti-TNF treatment (TC/aTNF and TC, respectively), and 6 week HRHF animals with or without anti-TNF treatment (6HRHF/aTNF and 6HRHF, respectively), in the last 2 weeks before euthanasia. ap<0.001 compared to NC; bp<0.05 compared to TC; cp<0.001 compared to TC; dp<0.001 compared to 6HRHF.
Figure 3
Figure 3. Anti-TNF intervention lowered PLF levels in forelimb bones and flexor digitorum muscles of high repetition high force (HRHF) task animals
A) Immunolocalization of PLF in forelimb bones (a–f) or flexor digitorum muscles (g–i) of 6 wk HRHF task animals with or without anti-TNF treatment. Sections were immunoreacted with PLF antibody; brown color indicates positive staining; hematoxylin (a nuclear stain) was used as a counterstain. PLF immunoreactivity was detected in radial periosteum (a–d) and osteoblasts (e–f, arrowheads), and flexor digitorum muscle fibers (g–h, arrowhead in g shows higher staining). PLF was also detected in macrophages (small arrow) in 6 wk HRHF task animals (i). (B,C) Total protein collected from radial/ulna bones (B) and flexor digitorum muscles (M) of normal controls (NC), 6 week HRHF animals with no anti-TNF treatment (B/HRHF and M/HRHF), and 6 week HRHF with anti-TNF treatment (B/ aTNF and M/ aTNF), and were resolved by 10% SDS PAGE and blots were probed with anti-PLF; GAPDH was used as a loading control. Experiments were repeated three times and bar graphs of the ratio of PLF/GAPDH are shown. a,b,cp<0.01 compared to NC, M/ aTNF and B/ aTNF, respectively. (D) Total protein collected from flexor digitorum muscles of normal control (NC), trained control with anti-TNF (TC/ aTNF), and 6 wk HRHF animals with or without anti-TNF (6HRHF/ aTNF and 6HRHF, respectively) was resolved by 10% SDS PAGE and probed with anti-CTGF antibody; GAPDH was used as a loading control. Experiments were repeated three times and bar graphs of the ratio of PLF/GAPDH are shown. (E) Western blot showing the single band for recombinant PLF made in E. coli, numbers 1–4 refer to the different clones examined for protein expression. Ps= Perisoteum; Ob= Osteoblast.
Figure 4
Figure 4. TNFα, MIP2 and MIP3a treatment increased PLF levels in primary osteoblasts culture in vitro
A–F) Primary osteoblasts were serum starved prior to treatment with TNFα, MIP2 or MIP3a at varying doses as indicated, or were untreated (C), then total protein was collected for western blot analysis 48 hrs after treatment. Total protein was resolved on 10% SDS-PAGE and blots probed with anti-PLF; GAPDH was used a loading control (A, C & E). Experiments were repeated three times and bar graphs of the ratio of PLF/GAPDH are shown (B, D & F). ap<0.01 compared to C; bp<0.01 compared to lower dose cytokine treatment.
Figure 5
Figure 5. PLF promotes primary osteoblast proliferation and increases CDK4 mRNA levels in primary osteoblast in vitro
A) Primary rat osteoblasts were infected with AdLacZ (expression of β galactosidase by adenovirus, control), AdPLF (over-expression of PLF by adenovirus), or AdsiPLF (adenoviral expression of small interfering RNA directed towards PLF, Knockdown) at a moi of 50. 72 h post-infection, proliferation was assessed by CyQUANT Cell Proliferation Assay Kit. Cell numbers were based on the fluorescence output (emission wavelength 520 nm). ap<0.05 compared to AdLacZ; bp<0.001 compared to AdsiPLF. B) Primary rat osteoblasts were treated with recombinant PLF (rPLF, 1–100ng/ml), recombinant Periostin (rPN, 100ng/ml), or were untreated. 48 h post-treatment, proliferation was assessed by CyQUANT Cell Proliferation Assay Kit. Cell numbers were based on the fluorescence output (emission wavelength 520 nm). Bar graphs represent fold change over untreated controls. a,b,cp<0.01 compared to 1 and 10 ng/ml rPLF, and rPN100, respectively. C) Primary rat osteoblasts were treated with recombinant PLF (rPLF, 1–100ng/ml), recombinant Periostin (rPN, 100ng/ml) or were untreated. 24 h post-treatment, BrdU was added to the cell cultures for additional 18 h and proliferation was assessed by ELISA performed on cell lysates and absorbance read at 495⌊. Bar graphs represent fold change over untreated control. a,b,cp<0.01 compared to 1 and 10 ng/ml rPLF, and rPN100, respectively. D) Primary rat osteoblasts were treated with recombinant PLF (100ng/ml) or were untreated. 24 h post-treatment, total RNA was collected and reverse transcribed to cDNA. Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) was used to assess the changes in CDK4 mRNA level between untreated controls and rPLF treated cultures. Experiments were repeated 3 times and values are expressed as fold change over control. Data was normalized using GAPDH mRNA levels. Right panel: Representative bar graph for pooled values from experiments 1–3.
Figure 6
Figure 6. PLF promotes MC3T3-E1 osteoblast-like cell adhesion and differentiation of primary osteoblast cells in vitro
A) MC3T3-E1 osteoblast-like cells were either left untreated or treated with anti-β1 integrin and plated in 96 well dishes coated with Poly-L-Lysine (PL; 0.10mg/ml), Fibronectin (FN; 5μg/ml), Periostin-like-factor conditioned media (PLF; 200μl/well) or green fluorescent protein conditioned media (GFP; 200μl/well) for 2 h at 37°C. Cells were fixed with 4% paraformaldehyde in PO4 buffer and stained with 1% methylene blue. Absorbance was read at 620⌊ on an ELISA plate reader. a,b,cp<0.01 compared to PL, GFP and GFP/β1, respectively. B) Primary rat osteoblasts were treated with recombinant PLF (100ng/ml), or were untreated. 48 h post-treatment, total RNA was collected and reverse transcribed to cDNA. Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) was used to assess the changes in RUNX2 mRNA levels between untreated controls and rPLF treated cultures. Experiments were repeated 3 times and values expressed as fold change over control data. Data was normalized using GAPDH mRNA levels. Right panel: Representative bar graph for pooled values from experiments 1–3. C) Primary rat osteoblasts were infected on day 2 of culture with AdLacZ (expression of β-galactosidase by adenovirus, control), AdPLF (over-expression of PLF by adenovirus), or AdsiPLF (adenoviral expression of siRNA directed towards PLF, knockdown) at a moi of 50. Cells were stained on day 7 for alkaline phosphatase (ALP); positive staining was red. D) ALP activity was assessed on parallel cultures, using p-nitrophenol as the substrate. a,bp<0.001 compared to AdLacZ and AdsiPLF, respectively. E) Primary rat osteoblasts were either untreated (Con) or treated with recombinant PLF (rPLF, 100ng/ml) or recombinant Periostin (rPN, 100ng/ml). Cells were stained on day 7 for ALP. F) ALP activity was assessed on parallel cultures. a,bp<0.001 and rPN, respectively. G) Rat primary osteoblasts were treated with recombinant PLF (100ng/ml), or were untreated. 48 h post-treatment, total RNA was collected and reverse transcribed to cDNA. Real Time Polymerase Chain Reaction (RT-PCR) was used to assess the changes in ALP mRNA levels between untreated controls and recombinant PLF treated cultures. Experiments were repeated 3 times and values expressed as fold change over controls. Data was normalized using GAPDH mRNA levels. Right panel: Representative bar graph for pooled values from experiments 1–3. H) Rat primary osteoblasts were treated with recombinant PLF (100ng/ml), or were untreated. 48 h post-treatment, total RNA was collected and reverse transcribed to cDNA. Real Time Polymerase Chain Reaction (RT-PCR) was used to assess the changes in osteocalcin (OC) mRNA level between untreated controls and recombinant PLF treated cultures. Experiments were repeated 3 times and values expressed as fold change over control. Data was normalized using GAPDH mRNA levels. Right panel: Representative bar graph for pooled values from experiments 1–3.
Figure 7
Figure 7. PLF over-expression or recombinant PLF enhanced calcium deposition and mineralization in rat primary osteoblasts on day 21 of culture
A–C) Primary rat osteoblasts were infected with AdLacZ (control), AdPLF (over-expression) or AdsiPLF (knockdown; siRNA against PLF) at a moi of 50. (A) Cells were stained with von Kossa for mineral deposition and counterstained with light green. (B) Bar graph of amount of calcium deposited per well. (C) Bar graph of average nodule area. a,bp<0.001 compared to AdLacZ and AdsiPLF, respectively. D–F) Rat primary osteoblasts were treated with recombinant PLF (100ng/ml), recombinant Perisotin (rPN-100ng/ml) or were untreated. (D) Cells were stained with von Kossa and light green. (E) Bar graph of amount of calcium deposited per well. (F) Bar graph of average nodule area. a,bp<0.001 and rPN, respectively.

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