Analysis of p38 MAP kinase pathway in osteoclasts

破骨细胞来源于单核/巨噬细胞系，负责骨吸收。P38MAPK是破骨细胞分化所必需的，但p38MAPK的特异性抑制剂SB203580不影响破骨细胞的存活和激活。骨吸收因子如IL-1和脂多糖(LPS)激活MAPK信号转导。为了阐明p38MAPK在破骨细胞功能中的作用，我们检测了MAPK的磷酸化状态，并检测了p38MAPK的强制磷酸化是否调节了破骨细胞的功能。尽管破骨细胞表达p38MAPK的水平与骨髓巨噬细胞相当，但脂多糖不能诱导破骨细胞p38MAPK的磷酸化。破骨细胞表达p38MAPK上游的MKK3和MKK6。IL-1和内毒素不能促进MKK3/6的磷酸化，甚至激活JNK和ERK。在破骨细胞中通过腺病毒表达活性形式的MKK6(MKK6CA)导致p38MAPK的磷酸化。48h时，表达MKK6CA的破骨细胞的存活活性与IL-1处理的细胞一样高。此外，经SB203580处理后，表达MKK6CA的成活破骨细胞数量减少到三分之一。表达MKK6CA的破骨细胞与对照组相比，牙本质片上的骨吸收活性和组织蛋白酶K的表达水平均无差异。这些发现表明，破骨细胞将自己的p38 MAPK信号保持在不活跃的状态，从而终止了自己的生存。在病理状态下，p38MAPK在破骨细胞中被激活，它可能延长破骨细胞的寿命。

Osteoclasts are derived from the monocyte/macrophage lineage and are responsible for bone resorption. p38 MAPK was essential for osteoclast differentiation but the survival and activation of osteoclasts were not affected by SB203580, a specific inhibitor for p38MAPK. Bone resorption factors such as IL-1 and lipopolysaccharide (LPS) activate MAPK signaling. To clarify the role of p38MAPK in osteoclast function, we measured the phosphorylated status of MAPKs and examined whether force-phosphorylation of p38MAPK modulates osteoclast function. Although osteoclasts expressed p38 MAPK at the level equivalent to that of bone marrow macrophages, phosphorylation of p38 MAPK was not induced by LPS in osteoclasts. MKK3 and MKK6, upstream kinases for p38 MAPK, were expressed in osteoclasts. Phosphorylation of MKK3/6 was not increased by IL-1 or LPS, even JNK and ERK were activated. Adenoviral expression of a constitutively active form of MKK6 (MKK6CA) in osteoclasts resulted in phosphorylation of p38 MAPK. The survival activity of MKK6CA-expressing osteoclasts evaluated at 48h was as high as that of IL-1-treated cells. Moreover, the number of survived osteoclasts which expressed MKK6CA decreased to a one third by SB203580. Neither bone resorbing activity on dentin slices nor the expression levels of cathepsin K were different between MKK6CA-expressing osteoclasts and control. These findings suggest that osteoclasts keep own p38 MAPK signaling in an inactive state to abort their survival. In a pathological state, p38 MAPK is activated in osteoclasts and it might extend osteoclast lifespan.