A novel regulatory role of TRAPPC9 in L‐plastin‐mediated osteoclast actin ring formation

Abstract

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF‐kB‐inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor‐κB (NF‐kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin‐K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC‐mediated cytoskeleton re‐organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L‐plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC‐mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL‐mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.

Plastin 3 in health and disease: a matter of balance

“…To analyze the proposed functions of PLS3 regarding mechanotransduction [5,15,108,120,143,144,148], Ca 2+ regulation [23,33,149], osteoblastic bone mineralization [15,120,126,131,134,143,144,150], osteoclastogenesis [16] and vesicular trafficking [15,16,61,151], various PLS3 mutations have been investigated and mouse model studies established to specifically investigate the effect of PLS3 loss or overexpression [16].…”

TRAPPC9 Modulation Regulates IL-1β-Induced Chondrocyte Inflammation

“…Trafficking Protein Particle Complex 9 (TRAPPC9) is a major subunit of the TRAP-PII Complex that has been shown to stimulate the NF-κB signaling cascade either through the canonical (through the IKKα/IKKβ/IKK-NEMO complex) or non-canonical (through NIK) pathway [25,76,77]. Furthermore, patients with TRAPPC9 frame-shift mutations present with both neuronal and skeletal abnormalities due to deleterious TRAPPC9-NF-κB signaling [9,34,63,78]. Despite these findings, no studies have examined the biological role of TRAPPC9 in chondrocytes or cartilage [33,36].…”

“…Next, we examined the mechanism by which TRAPPC9 enhances catabolic gene expression. Studies of TRAPPC9 signaling have demonstrated that TRAPPC9 (also known as NIK-and IKK2-binding protein (NIBP)) directly binds NIK and stimulates NF-κB signaling in neuronal, bone and cancer cells, where NF-κB activation maintains cancer cell malignancy and tumorigenesis [31,33,36,63,79]. Here, we provide the first evidence that TRAPPC9 binds NIK in primary murine chondrocytes, and that binding stimulates non-canonical NF-κB signaling through modulation of phospho-P-100, which has been shown to increase matrix catabolism and inflammation in OA [21][22][23] (Figure 4A, 5A-D).…”

“…TRAPPC9 has been shown to bind NF-κB inducing kinase (NIK/MAP3K14) in neuronal cells, cancer cells, and osteoclasts, but it is still unknown if this binding occurs in primary chondrocytes [31,35,36,63]. To test this interaction, primary murine articular chondrocytes were isolated from C57BL/6 male mice (n=5) and grown to confluency in culture.…”

The osteoclast cytoskeleton – current understanding and therapeutic perspectives for osteoporosis

“…L-fimbrin (also known as Lplastin) associates with osteoclast podosomes and with the actin patch of the nascent sealing zone (Chellaiah et al, 2018;Chellaiah et al, 2020). L-fimbrin recruitment to the actin patch requires phosphorylation by the leucine rich repeat kinase 1 (LRRK1) (Chellaiah et al, 2018;Si et al, 2018) and binding to the membrane-tethering complex protein TRAPPC9 (Hussein et al, 2019). Interestingly, mutations in LRRK1 give rise to a severe osteopetrotic phenotype in the mouse and cause human osteosclerotic metaphyseal dysplasia that is associated with osteoclast dysfunction (Miryounesi et al, 2019;Xing et al, 2013).…”