TRAPPC9 Analysis

Mutation in Trafficking Protein Particle Complex 9 (TRAPPC9) is linked to moderate to severe forms of NonSyndromic Autosomal Recessive Mental Retardation (NS-ARMRA), a disease which affects mostly patients of Middle-Eastern descent. Patients afflicted by TRAPPC9 mutations exhibit secondary phenotypes associated with musculoskeletal defects. TRAPPC9 is a protein subunit of Transport Protein Particle II318, which is localized at the ER exit sites.424 TRAPPC9 is required for protein trafficking that recycle through the early endosome.51 TRAPPC9 is also known to play a role during the transport of endoplasmic (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) through direct binding with p150Glued which plays an important
role in vesicle trafficking at the Golgi-ER exit site. TRAPPC9 gene is also known to encode the NIK – and IKK-β-binding protein (NIBP)167 which potentiates NF-kB activation through the direct interaction with NIK and IKKβ.
TRAPPC9 is highly expressed in muscle and kidney with relatively low expression in the brain, heart and placenta.167 The major known role of TRAPPC9 is associated with vesicles trafficking at the Golgi-ER exit site and its regulation of the NF-kB pathway through the direct interaction with NIK and IKK2 upstream.167 As a consequence, mutations in TRAPPC9 affect both vesicle transport51, 432, 433 and NF-kB activation.167 TRAPPC9 mutation is associated with NonSyndromic Autosomal Recessive Mental Retardation (NS-ARMR) in humans.
183, 237, 249, 252 TRAPPC9 knockdown study using siRNA showed deficiency in nerve growth factor-induced neuronal differentiation of PC12 cells through the attenuation of the TNFα-induced NF-κB activation and decreases the expression Bcl-xL gene.167 These studies underline the involvement of TRAPPC9 in NF-κB pathway activation during nervous system
development.
TRAPPC9 regulation of the NF-κB pathway is of particular interest to this study because numerous findings have underlined the role of NF-κB in bone cells differentiation and bone formation, suggesting that disruption of TRAPPC9 expression might also be relevant for bone homeostasis.
NF-κB is a family of nuclear factors involved in the regulation of many cell physiological and pathological processes.
NF-κB activation is known to be controlled through the canonical or classical 187 and non-canonical or alternate pathway333. The canonical activation is mediated through the translocation of the 65-kDa RelA and the 50-kDa NF-κB1 DNA-binding subunit, whereas, the non-canonical is thought to be mediated through the processing and translocation of the RelB complex. Emerging evidence shows that NF-κB activation negatively regulates osteoblast differentiation and function.7, 59 Inflammatory cytokines, in particular TNFα, provided the first indication of the inhibitor effect of the classical NF-κB pathway on bone formation in vivo and in vitro.129 The inhibitory effect of TNFα on osteoblast differentiation was mediated through Runx2 inhibition and was NF-kB dependent220. TNFα- and TNFR1- deficient mice showed increased basal bone mass in vivo and increased OB differentiation in vitro.220 Neil et al.7 showed that inhibition of NF-κB using S1627 NF-κB inhibitor increased osteoblast differentiation and bone formation through the up-regulation of osteoblast specific genes in vivo followed by an increased bone formation and repair bone defect in mouse calvarias critical defect model. The role on the non-classical NF-κB pathway in osteoblast was investigated by Chang et al.167 who showed that time and stage –specific inhibition of the IKK/NF-κB significantly increased
trabecular bone mass and bone mineralization and prevented ovariectomy bone loss in adult mice by maintaining bone formation in differentiated osteoblast. IKK/NF-κB inhibition showed enhanced JNK activation and increased Fra-1 expression.167 Furthermore, study of aly/aly mice showed that accumulation of the NF-κB2 precursor p100 enhanced osteoblast differentiation in vitro and bone formation in vivo.336
With mounting evidence of the role of NF-κB in osteoblast, we found that suppression of NF-κB activation owing to the regulation of TRAPPC9 greatly increases osteoblast differentiation in vitro and bone generation in vitro. Our data suggest that TRAPPC9 regulation affects osteoblast differentiation through NF-κB activation; TRAPPC9 down-regulation enhances osteoblast differentiation by up-regulation of different osteoblast marker such as Runx2 and ALP. In vivo experiment on critical size defect showed a greater recovery and bone generation in mice treated with MSCs transfected with TRAPPC9 shRNA. All together our data partially explain the bone phenotypes observed in patients suffering from NS-ARMR and for the first time provide evidence of the role of TRAPPC9 in bone.