Expression of Semaphorin-3A and its receptors in endochondral ossification: potential role in skeletal development and innervation.

Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin-3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin-3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin-3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin-3A signaling system (Semaphorin-3A, Neuropilin-1, and Plexins-A1 and -A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin-3A and Neuropilin-1 are also expressed in the bone marrow. Plexin-A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin-A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin-3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin-A3/Neuropilin-1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin-3A

Deficiency and Also Transgenic Overexpression of Timp-3 Both Lead to Compromised Bone Mass and Architecture In Vivo

Tissue inhibitor of metalloproteinases-3 (TIMP-3) regulates extracellular matrix via its inhibition of matrix metalloproteinases and membrane-bound sheddases. Timp-3 is expressed at multiple sites of extensive tissue remodelling. This extends to bone where its role, however, remains largely unresolved. In this study, we have used Micro-CT to assess bone mass and architecture, histological and histochemical evaluation to characterise the skeletal phenotype of Timp-3 KO mice and have complemented this by also examining similar indices in mice harbouring a Timp-3 transgene driven via a Col-2a-driven promoter to specifically target overexpression to chondrocytes. Our data show that Timp-3 deficiency compromises tibial bone mass and structure in both cortical and trabecular compartments, with corresponding increases in osteoclasts. Transgenic overexpression also generates defects in tibial structure predominantly in the cortical bone along the entire shaft without significant increases in osteoclasts. These alterations in cortical mass significantly compromise predicted tibial load-bearing resistance to torsion in both genotypes. Neither Timp-3 KO nor transgenic mouse growth plates are significantly affected. The impact of Timp-3 deficiency and of transgenic overexpression extends to produce modification in craniofacial bones of both endochondral and intramembranous origins. These data indicate that the levels of Timp-3 are crucial in the attainment of functionally-appropriate bone mass and architecture and that this arises from chondrogenic and osteogenic lineages

Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation

Diagenetic evolution of lower Jurassic platform carbonates flanking the Tazoult salt wall (Central High Atlas, Morocco)

Platform carbonates diagenesis in salt basins could be complex due to potential alterations of fluids related and non‐related to diapirism. This paper presents the diagenetic history of the Hettangian to Pliensbachian platform carbonates from the Tazoult salt wall area (central High Atlas, Morocco). Low structural relief and outcrop conditions allowed to define the entire diagenetic evolution occurred in the High Atlas diapiric basins since early stages of the diapiric activity up to their tectonic inversion. Precipitation of dolomite and calcite from both warmed marine‐derived and meteoric fluids characterised diagenetic stages during Pliensbachian, when the carbonate platforms were exposed and karstified. Burial diagenesis occurred from Toarcian to Middle Jurassic, due to changes of salt‐induced dynamic related to increase in siliciclastic input, fast diapir rise and rapid burial of Pliensbachian platforms. During this stage, the diapir acted as a physical barrier for fluid circulation between the core and the flanking sediments. In the carbonates and breccias flanking the structures, dolomite and calcite precipitated from basinal brines, whereas carbonate slivers located in the core of the structure, were affected by the circulation of Mn‐rich fluids. The final diagenetic event is characterised by the income of meteoric fluids into the system during uplift caused by Alpine orogeny. These results highlight the relevant influence of diapirism on the diagenetic modifications in salt‐related basins in terms of diagenetic events and involved fluids

Are Bone and Muscle Changes from POWER PE, an 8-month In-school Jumping Intervention, Maintained at Three Years?

Our aim was to determine if the musculoskeletal benefits of a twice-weekly, school-based, jumping regime in healthy adolescent boys and girls were maintained three years later. Subjects of the original POWER PE trial (n = 99) were contacted and asked to undergo retesting three years after cessation of the intervention. All original measures were completed including: sitting height, standing height, weight, calcaneal broadband ultrasound attenuation (BUA), whole body, hip and spine bone mineral content (BMC), lean tissue mass, and fat mass. Physical activity was recorded with the bone-specific physical activity questionnaire (BPAQ) and calcium intake was estimated with a calcium-focussed food questionnaire. Maturity was determined by Tanner staging and estimation of the age of peak height velocity (PHV). Twenty-nine adolescents aged 17.3±0.4 years agreed to participate. Three years after the intervention, there were no differences in subject characteristics between control and intervention groups (p>0.05). Three-year change in weight, lean mass, and fat mass were similar between groups (p>0.05). There were no significant group differences in three-year change in BUA or BMC at any site (p>0.05), although the between-group difference in femoral neck BMC at follow-up exceeded the least significant change. While significant group differences were not observed three years after cessation of the intervention, changes in bone parameters occurred in parallel for intervention and control groups such that the original benefits of the intervention observed within the treatment group were sustained

SNX12 Role in Endosome Membrane Transport

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes