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. 2013 Aug;114(8):1871-8.
doi: 10.1002/jcb.24531.

Zap70 inhibits Syk-mediated osteoclast function

Wei Zou  1 , Monica Croke, Tomohiro Fukunaga, Thomas J Broekelmann, Robert P Mecham, Steven L Teitelbaum
Affiliations

Zap70 inhibits Syk-mediated osteoclast function

Wei Zou et al. J Cell Biochem. 2013 Aug.

Abstract

The αvβ3 integrin stimulates the resorptive capacity of the differentiated osteoclast (OC) by organizing its cytoskeleton via the tyrosine kinase, Syk. Thus, Syk-deficient OCs fails to spread or form actin rings, in vitro and in vivo. The Syk family of tyrosine kinases consists of Syk itself and Zap70 which are expressed by different cell types. Because of their structural similarity, and its compensatory properties in other cells, we asked if Zap70 can substitute for absence of Syk in OCs. While expression of Syk, as expected, normalizes the cytoskeletal abnormalities of Syk(-/-) OCs, Zap70 fails do so. In keeping with this observation, Syk, but not Zap70, rescues αvβ3 integrin-induced SLP76 phosphorylation in Syk(-/-) OCs. Furthermore the kinase sequence of Syk partially rescues the Syk(-/-) phenotype but full normalization also requires its SH2 domains. Surprisingly, expression of Zap70 inhibits WT OC spreading, actin ring formation and bone resorptive activity, but not differentiation. In keeping with arrested cytoskeletal organization, Zap70 blocks integrin-activated endogenous Syk and Vav3, SLP76 phosphorylation. Such inhibition requires Zap70 kinase activity, as it is abolished by mutation of the Zap70 kinase domain. Thus, while the kinase domain of Syk is uniquely required for OC function that of Zap70 inhibits it.

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Conflict of interest statement

The authors declare they have no financial conflicts of interest.

Figures

Figure 1
Figure 1. Zap70 cannot rescue Syk-/- OCs function
A) WT macrophages, transduced with WT Zap70 or empty vector, were cultured with RANKL+ MCSF with time. Syk and Zap70 expression was determined by immunoblot. B) Syk-/- BMMs, transduced with Syk or Zap70, were cultured with RANKL and MCSF on plastic for 5 days. Cells were stained with TRAP activity (left panel) or FITC-phalloidin (right panel). C) Syk-/- BMMs, transduced with Syk or Zap70, were cultured with RANKL + MCSF, on bone, for 6 days. Cells were stained with FITC-phalloidin. D) Bone slices, described in C, were stained with peroxidase-labeled wheat germ agglutinin to visualize resorption lacunae after removal of osteoclasts. E) Syk-/- preosteoclasts, transduced with empty vector, Syk or Zap70, were maintained in suspension (S) or plated on vitronectin (A) for 30 minutes. SLP76 immunoprecipitates were immunoblotted for phosphorylated tyrosine (pY) and SLP76.
Figure 2
Figure 2. Syk kinase activity is essential for OC function
A) Scheme of Syk and Zap70 chimeric constructs. B) Syk-/- BMMs, transduced with Syk, Zap70 or chimeras, were cultured with RANKL and MCSF on plastic 5 days. Cells were stained with TRAP activity. C,D) Syk-/- BMMs, transduced with Syk, Zap70 or chimeras, were cultured with RANKL and MCSF on bone for 6 days. The cells were stained with FITC-phalloidin to visualize actin rings (C) or removed and the bone slices stained with peroxidase-labeled wheat germ agglutinin to delineate resorption lacunae (D).
Figure 3
Figure 3. Zap70 inhibits OC function via its kinase activity
A) WT BMMs, transduced with WT Syk, Zap70 or empty vector, were cultured with RANKL and MCSF on plastic (top panels) or bone slice (middle and lower panels) for 5-6 days. Cells were stained with TRAP activity (top panels) or FITC-phalloidin (middle panels). Resorption lacunae were visualized by peroxidase-labeled wheat germ agglutinin (lower panels). B) WT BMMs, transduced with WT Syk, Zap70 or empty vector, were cultured with RANKL and MCSF with time. Osteoclast differentiation markers were determined by immunoblot. C) WT BMMs, transduced with WT or kinase inactive (K369A) Zap70, were cultured with RANKL and MCSF on plastic for 5 days and stained for TRAP activity.
Figure 4
Figure 4. Zap70 does not affect Syk and DAP12 association
A) 293T cells were co-transfected with Dap12 and HA tagged Syk, WT or KD Zap70. HA immunoprecipitates were immunoblotted for DAP12 and HA. B) 293T cells were co-transfected with Flag-Dap12, Myc-Syk, and HA-WT or -KD Zap70. Flag immunoprecipitates were immunoblotted for Myc, HA and Flag.
Figure 5
Figure 5. Zap70 inhibits integrin-induced Syk activation
A-C) WT preosteoclasts, transduced with empty vector or WT Zap70, were maintained in suspension (S) or plated on vitronectin (A) for 30 minutes. A) Syk immunoprecipitates were immunoblotted for phosphorylated tyrosine (pY) and Syk. B) Vav3 immunoprecipitates were immunoblotted for phosphorylated tyrosine (pY) and Vav3. C) SLP76 immunoprecipitates were immunoblotted for phosphorylated tyrosine (pY) and SLP76. D) WT preosteoclasts, transduced with WT or kinase inactive Zap70 (K369A), were maintained in suspension (S) or plated on vitronectin (A) for 30 minutes. Syk immunoprecipitates were immunoblotted for phosphorylated tyrosine (pY) and Syk. E) WT preosteoclasts, transduced with empty vector, or WT Zap70, were maintained in suspension (S) or plated on vitronectin (A) for 30 minutes. Phospho-Src (p416) and c-Src were determined by immunoblot.
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