LIS1 Regulates Osteoclast Formation and Function through Its Interactions with Dynein/Dynactin and Plekhm1

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.

Activin A inhibits RANKL-mediated osteoclast formation, movement and function in murine bone marrow macrophage cultures

“…Bone marrow and marrow-derived macrophages were derived as previously described (Gaddy-Kurten et al, 2002;Ye et al, 2011). Briefly, the marrow cavities from femurs and tibias were flushed from mice with a-MEM containing 10% fetal bovine serum, antibiotics, and antimycotics (a-10 medium).…”

“…Cells were incubated at 37˚C in 5% CO 2 , 95% air for 3 days. Thereafter, the dishes were washed with PBS, and the adherent enriched population of mBMM cells were trypsinized and plated in BD Falcon tissue culture dishes to generate pre-OCLs and OCLs with 1/50th volume of CMG 14-12 culture supernatant and 50 ng/ml of recombinant mouse RANKL as previously described (Ye et al, 2011).…”

Dynein Light Chain LC8 Inhibits Osteoclast Differentiation and Prevents Bone Loss in Mice

“…Microtubule organization, intracellular vesicular trafficking, and the activity of dynein motor are critical for the activation and function of osteoclasts (46)(47)(48). LIS1 was recently identified as a microtubule regulator that interacts with both Plekhm1, a component of late endosomal trafficking, and the dynein-dynactin motor complex in osteoclasts (47).…”

“…LIS1 was recently identified as a microtubule regulator that interacts with both Plekhm1, a component of late endosomal trafficking, and the dynein-dynactin motor complex in osteoclasts (47). Depletion of LIS1 dramatically inhibited the formation and bone resorption function of osteoclasts by interfering with dynein function and microtubule organization (47). Exogenous expression of dynamitin, a component of the dynactin complex, disrupted the dynein-dynactin complex, leading to retardation of osteoclast formation (48).…”

Regulation of lysosome biogenesis and functions in osteoclasts

“…This study and more recent work implicating the dynein-dynactin complex proteins and LIS1, a microtubule regulator in the resorptive capacity of osteoclasts, suggest that None the active transport of vesicular cargo along microtubules is essential to osteoclasts function. 68,69 Moreover, nocodazole, a microtubule-depolymerizing reagent, inhibits the bone resorptive activity of differentiated osteoclasts, 70 illustrating the importance of microtubule networks in osteoclast function.…”