Abstract
Microtubule organization and lysosomal secretion are both critical for the activation and function of osteoclasts, highly specialized polykaryons that are responsible for bone resorption and skeletal homeostasis. Here, we have identified a novel interaction between microtubule regulator LIS1 and Plekhm1, a lysosome-associated protein implicated in osteoclast secretion. Decreasing LIS1 expression by shRNA dramatically attenuated osteoclast formation and function, as shown by a decreased number of mature osteoclasts differentiated from bone marrow macrophages, diminished resorption pits formation, and reduced level of CTx-I, a bone resorption marker. The ablated osteoclast formation in LIS1-depleted macrophages was associated with a significant decrease in macrophage proliferation, osteoclast survival and differentiation, which were caused by reduced activation of ERK and AKT by M-CSF, prolonged RANKL-induced JNK activation and declined expression of NFAT-c1, a master transcription factor of osteoclast differentiation. Consistent with its critical role in microtubule organization and dynein function in other cell types, we found that LIS1 binds to and colocalizes with dynein in osteoclasts. Loss of LIS1 led to disorganized microtubules and aberrant dynein function. More importantly, the depletion of LIS1 in osteoclasts inhibited the secretion of Cathepsin K, a crucial lysosomal hydrolase for bone degradation, and reduced the motility of osteoclast precursors. These results indicate that LIS1 is a previously unrecognized regulator of osteoclast formation, microtubule organization, and lysosomal secretion by virtue of its ability to modulate dynein function and Plekhm1.
Highlights
Osteoclasts are terminally differentiated polykaryons that are uniquely capable of digesting calcified bone matrix
To further map which part(s) of Plekhm1 interact with LIS1, we retrovirally transduced a series of truncated fragments of murine Plekhm1 into bone marrow macrophages (BMMs) which were further cultured with macrophage colonystimulating factor (M-CSF) and RANKL for 5 days to generate osteoclasts (Figure 1D)
While immunoprecipitated endogenous LIS1 was associated with the C-terminal half of Plekhm1, deletion of the RUN and/or PH1 domains abolished Plekhm1-LIS1 interaction (Figure 1D)
Summary
Osteoclasts are terminally differentiated polykaryons that are uniquely capable of digesting calcified bone matrix. They are formed by fusion of mononuclear precursors of the monocyte/ macrophage lineage [1,2]. NFATc1 is induced by RANKL and co-activated by immunoglobulin-like receptors and their associated adapter proteins [4,5]. As they mature, osteoclasts undergo dramatic reorganization of their cytoskeleton. When osteoclasts are cultured on glass or plastic, individual podosomes are clustered and expand to the cell periphery to form a stable ‘‘podosome belt’’
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