Identification of receptor-type protein tyrosine phosphatase µ as a new marker for osteocytes.

Osteocytes are the predominant cells in bone, where they form a cellular network and display important functions in bone homeostasis, phosphate metabolism and mechanical transduction. Several proteins strongly expressed by osteocytes are involved in these processes, e.g., sclerostin, DMP-1, PHEX, FGF23 and MEPE, while others are upregulated during differentiation of osteoblasts into osteocytes, e.g., osteocalcin and E11. The receptor-type protein tyrosine phosphatase µ (RPTPµ) has been described to be expressed in cells which display a cellular network, e.g., endothelial and neuronal cells, and is implied in mechanotransduction. In a capillary outgrowth assay using metatarsals derived from RPTPµ-knock-out/LacZ knock-in mice, we observed that the capillary structures grown out of the metatarsals were stained blue, as expected. Surprisingly, cells within the metatarsal bone tissue were positive for LacZ activity as well, indicating that RPTPµ is also expressed by osteocytes. Subsequent histochemical analysis showed that within bone, RPTPµ is expressed exclusively in early-stage osteocytes. Analysis of bone marrow cell cultures revealed that osteocytes are present in the nodules and an enzymatic assay enabled the quantification of the amount of osteocytes. No apparent bone phenotype was observed when tibiae of RPTPµ-knock-out/LacZ knock-in mice were analyzed by µCT at several time points during aging, although a significant reduction in cortical bone was observed in RPTPµ-knock-out/LacZ knock-in mice at 20 weeks. Changes in trabecular bone were more subtle. Our data show that RPTPµ is a new marker for osteocytes.

Downregulation of platelet responsiveness upon contact with LDL by the protein-tyrosine phosphatases SHP-1 and SHP-2.

OBJECTIVE: The sensitivity of platelets to aggregating agents increases when low-density lipoprotein (LDL) binds to apolipoprotein E receptor 2' (apoER2'), triggering activation of p38MAPK and formation of thromboxane A2. LDL signaling is terminated by PECAM-1 through recruitment and activation of the Ser/Thr protein phosphatase PP2A, but platelets remain unresponsive to LDL when PECAM-1 activation disappears. We report a second mechanism that halts LDL signaling and in addition lowers platelet responsiveness to aggregating agents. METHODS AND RESULTS: After a first stimulation with LDL, platelets remain unresponsive to LDL for 60 minutes, despite normal apoER2' activation by a second dose of LDL. A possible cause is persistent activation of the tyrosine phosphatases SHP-1 and SHP-2, which may not only block a second activation of p38MAPK, PECAM-1, and PP2A by LDL but also seem to reduce aggregation by TRAP, collagen, and ADP. CONCLUSION: These findings reveal that p38MAPK phosphorylation and platelet activation by LDL are suppressed by two mechanisms: (1) short activation of PECAM-1/PP2A, and (2) prolonged activation of SHP-1 and SHP-2. Activation of SHP-1 and SHP-2 is accompanied by reduced responsiveness to aggregating agents, which--if present in vivo--would make LDL an aggregation inhibitor during prolonged contact with platelets.

Binding of low density lipoprotein to platelet apolipoprotein E receptor 2' results in phosphorylation of p38MAPK.

Binding of low density lipoprotein (LDL) to platelets enhances platelet responsiveness to various aggregation-inducing agents. However, the identity of the platelet surface receptor for LDL is unknown. We have previously reported that binding of the LDL component apolipoprotein B100 to platelets induces rapid phosphorylation of p38 mitogen-activated protein kinase (p38MAPK). Here, we show that LDL-dependent activation of this kinase is inhibited by receptor-associated protein (RAP), an inhibitor of members of the LDL receptor family. Confocal microscopy revealed a high degree of co-localization of LDL and a splice variant of the LDL receptor family member apolipoprotein E receptor-2 (apoER2') at the platelet surface, suggesting that apoER2' may contribute to LDL-induced platelet signaling. Indeed, LDL was unable to induce p38MAPK activation in platelets of apoER2-deficient mice. Furthermore, LDL bound efficiently to soluble apoER2', and the transient LDL-induced activation of p38MAPK was mimicked by an anti-apoER2 antibody. Association of LDL to platelets resulted in tyrosine phosphorylation of apoER2', a process that was inhibited in the presence of PP1, an inhibitor of Src-like tyrosine kinases. Moreover, phosphorylated but not native apoER2' co-precipitated with the Src family member Fgr. This suggests that exposure of platelets to LDL induces association of apoER2' to Fgr, a kinase that is able to activate p38MAPK. In conclusion, our data indicate that apoER2' contributes to LDL-dependent sensitization of platelets.