SH3BP2 is an activator of NFAT activity and osteoclastogenesis

Abstract

Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Osteoclasts differentiate after binding of sRANKL to RANK induces a number of downstream signaling effects, including activation of the calcineurin/NFAT (nuclear factor of activated T cells) pathway. Here, we have investigated the functional significance of SH3BP2 protein on osteoclastogenesis in the presence of sRANKL. Our results indicate that SH3BP2 both increases nuclear NFATc1 in sRANKL treated RAW 264.7 preosteoclast cells and enhances expression of tartrate resistant acid phosphatase (TRAP), a specific marker of osteoclast differentiation. Moreover, overexpression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLCgamma1 and 2, thus providing a mechanistic pathway for the rapid translocation of NFATc1 into the nucleus and increased osteoclastogenesis in cherubism.

FIG. 1

A SH3BP2 INCREASES THE NUCLEAR TRANSLOCATION OF NFATC1. RAW 264.7 cells were transiently transfected with murine SH3BP2 (red) and NFATc1 (green) translocated to the nucleus (A) compared to cells not transfected with SH3BP2 (B). SH3BP2 did not increase the total amount of NFATc1 in cells however SH3BP2 with sRANKL (100ng/ml) increased NFATc1 activation by increasing the translocation. C. SH3BP2 INCREASES NFAT ACTIVITY. The figure demonstrates that in RAW 264.7 cells stimulated with sRANKL (100ng/ml), murine SH3BP2 cDNA significantly increases NFAT activity compared to the pMT3 vector only (p<0.0003), sRANKL (p<0.0001) or SH3BP2 alone (p<0.0001). This experiment was repeated four times with similar results. D. TRAP ACTIVITY IS INCREASED IN THE PRESENCE OF SH3BP2. RAW 264.7 cells were transiently transfected with murine SH3BP2 and stimulated with sRANKL (100ng/ml). In RANKL stimulated (100ng/ml) and SH3BP2 transfected cells TRAP activity was significantly increased compared to the vector only (pMT3 vector) control p<0.0001, SH3BP2 alone (p<0.0001) and sRANKL stimulation alone (p=0.001). # indicates p<0.02 and ## indicates p<0.0001. This experiment was repeated three times with similar results.

FIG. 1

A SH3BP2 INCREASES THE NUCLEAR TRANSLOCATION OF NFATC1. RAW 264.7 cells were transiently transfected with murine SH3BP2 (red) and NFATc1 (green) translocated to the nucleus (A) compared to cells not transfected with SH3BP2 (B). SH3BP2 did not increase the total amount of NFATc1 in cells however SH3BP2 with sRANKL (100ng/ml) increased NFATc1 activation by increasing the translocation. C. SH3BP2 INCREASES NFAT ACTIVITY. The figure demonstrates that in RAW 264.7 cells stimulated with sRANKL (100ng/ml), murine SH3BP2 cDNA significantly increases NFAT activity compared to the pMT3 vector only (p<0.0003), sRANKL (p<0.0001) or SH3BP2 alone (p<0.0001). This experiment was repeated four times with similar results. D. TRAP ACTIVITY IS INCREASED IN THE PRESENCE OF SH3BP2. RAW 264.7 cells were transiently transfected with murine SH3BP2 and stimulated with sRANKL (100ng/ml). In RANKL stimulated (100ng/ml) and SH3BP2 transfected cells TRAP activity was significantly increased compared to the vector only (pMT3 vector) control p<0.0001, SH3BP2 alone (p<0.0001) and sRANKL stimulation alone (p=0.001). # indicates p<0.02 and ## indicates p<0.0001. This experiment was repeated three times with similar results.

FIG. 1

A SH3BP2 INCREASES THE NUCLEAR TRANSLOCATION OF NFATC1. RAW 264.7 cells were transiently transfected with murine SH3BP2 (red) and NFATc1 (green) translocated to the nucleus (A) compared to cells not transfected with SH3BP2 (B). SH3BP2 did not increase the total amount of NFATc1 in cells however SH3BP2 with sRANKL (100ng/ml) increased NFATc1 activation by increasing the translocation. C. SH3BP2 INCREASES NFAT ACTIVITY. The figure demonstrates that in RAW 264.7 cells stimulated with sRANKL (100ng/ml), murine SH3BP2 cDNA significantly increases NFAT activity compared to the pMT3 vector only (p<0.0003), sRANKL (p<0.0001) or SH3BP2 alone (p<0.0001). This experiment was repeated four times with similar results. D. TRAP ACTIVITY IS INCREASED IN THE PRESENCE OF SH3BP2. RAW 264.7 cells were transiently transfected with murine SH3BP2 and stimulated with sRANKL (100ng/ml). In RANKL stimulated (100ng/ml) and SH3BP2 transfected cells TRAP activity was significantly increased compared to the vector only (pMT3 vector) control p<0.0001, SH3BP2 alone (p<0.0001) and sRANKL stimulation alone (p=0.001). # indicates p<0.02 and ## indicates p<0.0001. This experiment was repeated three times with similar results.

FIG. 2

A. CYCLOSPORINE A INHIBITS SH3BP2/sRANKL INDUCED NFAT ACTIVITY. RAW 264.7, transiently transfected with murine SH3BP2 and stimulated with sRANKL (100ng/ml), were treated with Cyclosporine A. The calcineurin inhibitor (cyclosporine A) inhibits SH3BP2/sRANKL indicating that SH3BP2 is acting upstream of calcineurin. ## indicates p< 0.0001. B. The PI-PLC inhibitor (U-73122) has a mild inhibitory effect on NFAT activation. RAW 264.7 cells were transiently transfected with murine SH3BP2, stimulated with RANKL and treated with U-73122 or U-77343 (the inactive analog of U-73122)[23] The bar graph demonstrates an inhibition of NFAT activity with U-73122 at a dose of 3μM compared to U-73343. # indicates p<0.04.

FIG. 2

A. CYCLOSPORINE A INHIBITS SH3BP2/sRANKL INDUCED NFAT ACTIVITY. RAW 264.7, transiently transfected with murine SH3BP2 and stimulated with sRANKL (100ng/ml), were treated with Cyclosporine A. The calcineurin inhibitor (cyclosporine A) inhibits SH3BP2/sRANKL indicating that SH3BP2 is acting upstream of calcineurin. ## indicates p< 0.0001. B. The PI-PLC inhibitor (U-73122) has a mild inhibitory effect on NFAT activation. RAW 264.7 cells were transiently transfected with murine SH3BP2, stimulated with RANKL and treated with U-73122 or U-77343 (the inactive analog of U-73122)[23] The bar graph demonstrates an inhibition of NFAT activity with U-73122 at a dose of 3μM compared to U-73343. # indicates p<0.04.

FIG. 3

A. 2-APB inhibits NFAT activation. 2-APB, the known IP3R inhibitor, inhibits NFAT activity in all groups (with or without murine SH3BP2 and/or sRANKL) in RAW 264.7 cells (# indicates p<0.02 and ## indicates p<0.0001). B. TRAP staining and TRAP activity were nearly eliminated by 2-APB. 2-APB inhibited TRAP activity in RAW 264.7 cells after one week of culture indicating an inhibition of osteoclastogenesis. # indicates p<0.02 and ## indicates p<0.005.

FIG. 3

A. 2-APB inhibits NFAT activation. 2-APB, the known IP3R inhibitor, inhibits NFAT activity in all groups (with or without murine SH3BP2 and/or sRANKL) in RAW 264.7 cells (# indicates p<0.02 and ## indicates p<0.0001). B. TRAP staining and TRAP activity were nearly eliminated by 2-APB. 2-APB inhibited TRAP activity in RAW 264.7 cells after one week of culture indicating an inhibition of osteoclastogenesis. # indicates p<0.02 and ## indicates p<0.005.

FIG. 4

SH3BP2 INCREASES PHOSPHORYLATION OF PLCγ2 AND SYK. A. The figure demonstrates that SH3BP2 increases the phosphorylation but not the total amount of Syk. B. The figure demonstrates that SH3BP2 increases the PLCγ2 phosphorylation but not the total amount of PLCγ2. β-actin was used as a control to ensure that equal protein was loaded in each lane. This experiment was repeated three times with similar results. VO represents cells transiently transfected with vector only and Mo represents cells transiently transfected with murine SH3BP2.

FIG. 4

SH3BP2 INCREASES PHOSPHORYLATION OF PLCγ2 AND SYK. A. The figure demonstrates that SH3BP2 increases the phosphorylation but not the total amount of Syk. B. The figure demonstrates that SH3BP2 increases the PLCγ2 phosphorylation but not the total amount of PLCγ2. β-actin was used as a control to ensure that equal protein was loaded in each lane. This experiment was repeated three times with similar results. VO represents cells transiently transfected with vector only and Mo represents cells transiently transfected with murine SH3BP2.