Targeted Ptpn11 deletion in mice reveals the essential role of SHP2 in osteoblast differentiation and skeletal homeostasis

The maturation and function of osteoblasts(OBs)rely heavily on the reversible phosphorylation of signaling proteins.To date,most of the work in OBs has focused on phosphorylation by tyrosyl kinases,but little has been revealed about dephosphorylation by protein tyrosine phosphatases(PTPases).SHP2(encoded by PTPN11)is a ubiquitously expressed PTPase.PTPN11 mutations are associated with both bone and cartilage manifestations in patients with Noonan syndrome(NS)and metachondromatosis(MC),although the underlying mechanisms remain elusive.Here,we report that SHP2 deletion in bone gamma-carboxyglutamate protein-expressing(Bglap+)bone cells leads to massive osteopenia in both trabecular and cortical bones due to the failure of bone cell maturation and enhanced osteoclast activity,and its deletion in Bglap+chondrocytes results in the onset of enchondroma and osteochondroma in aged mice with increased tubular bone length.Mechanistically,SHP2 was found to be required for osteoblastic differentiation by promoting RUNX2/OSTERIX signaling and for the suppression of osteoclastogenesis by inhibiting STAT3-mediated RANKL production by osteoblasts and osteocytes.These findings are likely to explain the compromised skeletal system in NS and MC patients and to inform the development of novel therapeutics to combat skeletal disorders.

A multiscale 3D finite element analysis of fluid/solute transport in mechanically loaded bone

The transport of fluid, nutrients, and signaling molecules in the bone lacunar-canalicular system （LCS） is critical for osteocyte survival and function. We have applied the fluorescence recovery after photobleaching （FRAP） approach to quantify load-induced fluid and solute transport in the LCS in situ, but the measurements were limited to cortical regions 30-50 μm underneath the periosteum due to the constrains of laser penetration. With this work, we aimed to expand our understanding of load-induced fluid and solute transport in both trabecular and cortical bone using a multiscaled image-based finite element analysis （FEA） approach. An intact murine tibia was first re-constructed from microCT images into a three-dimensional （3D） linear elastic FEA model, and the matrix deformations at various locations were calculated under axial loading. A segment of the above 3D model was then imported to the biphasic poroelasticity analysis platform （FEBio） to predict load-induced fluid pressure fields, and interstitial solute/fluid flows through LCS in both cortical and trabecular regions. Further, secondary flow effects such as the shear stress and/or drag force acting on osteocytes, the presumed mechano-sensors in bone, were derived using the previously developed ultrastructural model of Brinkman flow in the canaliculi. The material properties assumed in the FEA models were validated against previously obtained strain and FRAP transport data measured on the cortical cortex. Our results demonstrated the feasibility of this computational approach in estimating the fluid flux in the LCS and the cellular stimulation forces （shear and drag forces） for osteocytes in any cortical and trabecular bone locations, allowing further studies of how the activation of osteocytes correlates with in vivo functional bone formation. The study provides a promising platform to reveal potential cellular mechanisms underlying the anabolic power of exercises and physical activities in treating patients with skeletal deficiencies.

Perlecan/Hspg2 Deficiency Impairs Bone’s Calcium Signaling and Associated Transcriptome in Response to Mechanical Loading

Perlecan,a heparan sulfate proteoglycan,acts as a mechanical sensor for bone to detect external loading.Deficiency of perlecan increases the risk of osteoporosis in patients with Schwartz-Jampel Syndrome(SJS)and attenuates loading4nduced bone formation in perlecan deficient mice(Hypo).Considering that intracellular calcium[Ca2+]i is an ubiquitous messenger controlling numerous cellular processes including mechanotransduction,we hypothesized that perlecan deficiency impairs bone’s calcium signaling in response to loading.To test this,we performed real-time[Ca2+]i imaging on in situ osteocytes of adult murine tibiae under cyclic loading(8 N,Figure 1).Relative to wild type(WT),Hypo osteocytes showed decreases in the overall[Ca2+]i response rate(-58%),calcium peaks(-33%),cells with multiple peaks(-53%),peak magnitude(-6.8%),and recovery speed to baseline(-23%).RNA sequencing and pathway analysis of tibiae from mice subjected to one or seven days of unilateral loading demonstrated that perlecan deficiency significantly suppressed the calcium signaling,ECM-receptor interaction,and focal adhesion pathways following repetitive loading.Defects in the endoplasmic reticulum(ER)calcium cycling regulators such as Ryr1/ryanodine receptors and Atp2a1/Sercal calcium pumps were identified in Hypo bones.Taken together,impaired calcium signaling may contribute to bone’s reduced anabolic response to loading,underlying the osteoporosis risk for the SJS patients.