E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models

Abstract

The transmembrane glycoprotein E11 is considered critical in early osteoblast–osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO‐A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post‐translational degradation. We found that exogenous treatment of MLO‐A5 and osteocytic IDG‐SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome‐selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin‐A) produced similar dose‐dependent increases in E11 protein levels in MLO‐A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO‐A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome‐mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. J. Cell. Physiol. 231: 1392–1404, 2016. © 2015 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.

Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation

“…Conversely, the ectopic overexpression of Pdpn in keratinocytes induces plasma membrane extensions (Scholl, Gamallo, Vilaro, & Quintanilla, ), and in endothelial cells, the reorganization of the actin cytoskeleton and the formation of long tube like structures (Schacht et al, ). Similarly, we have previously shown that stabilization of Pdpn protein, through inhibition of endogenous proteasome activity, promotes dendrite formation through the RhoA/ROCK/ERM pathway (Staines, Prideaux, et al, ). RhoA is a small GTPase and a master regulator of various cellular processes such as cytokinetics, cytoskeletal regulation, and cell migration (Takai, Sasaki, & Matozaki, ).…”

“…We, and others, have previously shown that Pdpn promotes osteocytogenesis and dendrite formation in vitro (Staines, Prideaux, et al, ; Zhang et al, ). Our studies, described herein, have for the first time successfully generated a bone‐specific conditional Pdpn hypomorphic mouse and we have confirmed a role for Pdpn in the attainment of fully elongated osteocyte dendrites.…”

“…We have previously shown that Pdpn is expressed by early embedding osteocytes, thus identifying it as a factor which likely contributes to the vital, early stages of osteocyte differentiation (Staines, Prideaux, et al, ). It is known that Pdpn expression in osteocytes is up‐regulated in response to mechanical strain in vivo (Zhang et al, ) and that increased Pdpn expression, through overexpression in ROS 17/2.6 cells and through stabilization by proteasome inhibitors in MLO‐A5 cells, leads to the formation of long dendritic processes (Sprague, Wetterwald, Heinzman, & Atkinson, ; Staines, Prideaux, et al, ). The formation of these cytoplasmic processes is abrogated in cells pre‐treated with siRNA targeted against Pdpn (Zhang et al, ).…”

Conditional deletion of E11/Podoplanin in bone protects against ovariectomy-induced increases in osteoclast formation and activity

“…We, and others, have previously highlighted the requirement for the transmembrane glycoprotein E11/podoplanin (Pdpn) to make the cytoskeletal network modifications that are critical for the formation of dendrites during osteocytogenesis. This role is consistent with its expression by early embedding osteocytes, thus identifying Pdpn as a factor which likely contributes to the vital, early stages of osteocyte differentiation [11]. Furthermore, Pdpn expression in osteocytes is increased in response to mechanical strain in vivo [12] and the formation of dendritic processes is promoted by both Pdpn overexpression and through its stabilisation by proteasome inhibitors [11,13].…”

CLEC-2 suppresses calcification in cultured osteoblasts

“…Differentiation of cultured mouse calvarial osteoblasts and pre-osteoblastoid cell MLO-A5 into osteocytes in calcification medium containing βglycerophosphate and ascorbic acid, and biomaterial-induced calcification are simultaneously accompanied by an increase in podoplanin (24)(25)(26)(27). Also, in the mature osteoblast MLO-A5 cell line, an increase in podoplanin coincides with dendrite formation, calcification, and RhoA activation (28).…”