Bone loss recovery in mice following microgravity with concurrent bone-compartment-specific osteocyte characteristics.

Space missions provide the opportunity to investigate the influence of gravity on the dynamic remodelling processes in bone. Mice were examined following space flight and subsequent recovery to determine the effects on bone compartment-specific microstructure and composition. The resulting bone loss following microgravity recovered only in trabecular bone, while in cortical bone the tissue mineral density was restored after only one week on Earth. Detection of TRAP-positive bone surface cells in the trabecular compartment indicated increased resorption following space flight. In cortical bone, a persistent reduced viability of osteocytes suggested an impaired sensitivity to mechanical stresses. A compartment-dependent structural recovery from microgravity-induced bone loss was shown, with a direct osteocytic contribution to persistent low bone volume in the cortical region even after a recovery period. Trabecular recovery was not accompanied by changes in osteocyte characteristics. These post-space-flight findings will contribute to the understanding of compositional changes that compromise bone quality caused by unloading, immobilisation, or disuse.

Skeletal deterioration in COL2A1-related spondyloepiphyseal dysplasia occurs prior to osteoarthritis.

OBJECTIVE: Spondyloepiphyseal dysplasia, a combination of progressive arthropathy with variable signs of skeletal dysplasia, can be a result of mutations in the collagen, type II, alpha 1 (COL2A1) gene. However, the bone involvement (e.g., density, microstructure) in this disorder has hitherto not been studied. DESIGN: A 50-year-old female patient and her 8-year-old son with flattening of vertebral bodies and early-onset osteoarthritis were genetically tested using a custom designed gene bone panel including 386 genes. Bone microstructure and turnover were assessed using high-resolution peripheral quantitative computed tomography (HR-pQCT) and serum bone turnover markers, respectively. Furthermore, the bone and cartilage phenotype of male mice heterozygous for the loss-of-function mutation of Col2a1 (Col2a1+/d) was analyzed compared to wildtype littermates using µ-CT and histomorphometry. RESULTS: We identified a dominant COL2A1 mutation (c.620G > A p.(Gly207Glu)) indicating spondyloepiphyseal dysplasia in the female patient and her son, both being severely affected by skeletal deterioration. Although there was no osteoarthritis detectable at first visit, the son was affected by trabecular osteopenia, which progressed over time. In an iliac crest biopsy obtained from the mother, osteoclast indices were remarkably increased. Col2a1+/d mice developed a moderate skeletal phenotype expressed by reduced cortical and trabecular parameters at 4 weeks. Importantly, no articular defects could be observed in the knee joints at 4 weeks, while osteoarthritis was only detectable in 12-week-old mice. CONCLUSIONS: Our results indicate that collagen type II deficiency in spondyloepiphyseal dysplasia leads to skeletal deterioration with early-onset in humans and mice that occurs prior to the development of osteoarthritis.

A novel FAM20C mutation causing hypophosphatemic osteomalacia with osteosclerosis (mild Raine syndrome) in an elderly man with spontaneous osteonecrosis of the knee.

Raine syndrome is characterized by FGF23-mediated hypophosphatemic osteomalacia with osteosclerosis caused by mutations in the FAM20C gene. We report a case of a 72-year-old man who presented with rapid progressive spontaneous osteonecrosis of the knee (SONK). A full osteologic assessment including dual energy X-ray absorptiometry (DXA), high-resolution peripheral quantitative computed tomography (HR-pQCT), and serum analyses revealed a high bone mass in the lumbar spine and hip (DXA T-score + 7.5 and + 4.7/+4.2) with increased bone microstructural parameters in the distal radius and tibia (BV/TV 127%, 140% of the age-matched mean, respectively), as well as a low bone turnover state. Phosphate levels were low due to renal phosphate wasting and high FGF23 levels (126.5 pg/ml, reference range 23.2-95.4 pg/ml). Using gene panel sequencing, we identified a novel FAM20C heterozygous missense mutation in combination with a homozygous duplication that potentially alters splicing. Taken together, this is the first case of mild Raine syndrome with spontaneous osteonecrosis of the knee, phosphate wasting, and a pronounced trabecular high bone mass phenotype.

A novel COL1A2 C-propeptide cleavage site mutation causing high bone mass osteogenesis imperfecta with a regional distribution pattern.

Osteogenesis imperfecta (OI) is typically characterized by low bone mass and increased bone fragility caused by heterozygous mutations in the type I procollagen genes (COL1A1/COL1A2). We report two cases of a 56-year-old woman and her 80-year-old mother who suffered from multiple vertebral and non-vertebral fractures with onset in early childhood. A full osteologic assessment including dual-energy X-ray absorptiometry (DXA), high-resolution peripheral quantitative computed tomography (HR-pQCT), and serum analyses pointed to a high bone mineral density (BMD) in the hip (DXA Z-score + 3.7 and + 3.9) but low to normal bone mass in the spine and preserved bone microstructure in the distal tibia. Serum markers of bone formation and bone resorption were elevated. Using whole exome sequencing, we identified a novel mutation in the COL1A2 gene causing a p. (Asp1120Gly) substitution at the protein level and affecting the type I procollagen C-propeptide cleavage site. In line with previously reported cases, our data independently prove the existence of an unusual phenotype of high bone mass OI caused by a mutation in the procollagen C-propeptide cleavage with a clinically persistent phenotype through adulthood.

Mutational analysis uncovers monogenic bone disorders in women with pregnancy-associated osteoporosis: three novel mutations in LRP5, COL1A1, and COL1A2.

Pregnancy was found to be a skeletal risk factor promoting the initial onset of previously unrecognized monogenic bone disorders, thus explaining a proportion of cases with pregnancy-associated osteoporosis. Therapeutic measures should focus in particular on the normalization of the disturbed calcium homeostasis in order to enable the partial skeletal recovery. INTRODUCTION: Pregnancy-associated osteoporosis (PAO) is a rare skeletal condition, which is characterized by a reduction in bone mineral density (BMD) in the course of pregnancy and lactation. Typical symptoms include vertebral compression fractures and transient osteoporosis of the hip. Since the etiology is not well understood, this prospective study was conducted in order to elucidate the relevance of pathogenic gene variants for the development of PAO. METHODS: Seven consecutive cases with the diagnosis of PAO underwent a skeletal assessment (blood tests, DXA, HR-pQCT) and a comprehensive genetic analysis using a custom-designed gene panel. RESULTS: All cases showed a reduced BMD (DXA T-score, lumbar spine - 3.2 ± 1.0; left femur - 2.2 ± 0.5; right femur - 1.9 ± 0.5), while the spine was affected more severely (p < 0.05). The trabecular and cortical thickness was overall reduced in HR-pQCT, while the trabecular number showed no alterations in most cases. The genetic analysis revealed three novel mutations in LRP5, COL1A1, and COL1A2. CONCLUSION: Our data show that previously unrecognized monogenic bone disorders play an important role in PAO. Pregnancy should be considered a skeletal risk factor, which can promote the initial clinical onset of such skeletal disorders. The underlying increased calcium demand is essential in terms of prophylactic and therapeutic measures, which are especially required in individuals with a genetically determined low bone mass. The implementation of this knowledge in clinical practice can enable the partial recovery of the skeleton. Consistent genetic studies are needed to analyze the frequency of pathogenic variants in women with PAO.

Calcium gluconate supplementation is effective to balance calcium homeostasis in patients with gastrectomy.

UNLABELLED: We demonstrate histological evidence for hyperparathyroidism in patients with gastrectomy. This is, at least in part, explained by impaired calcium absorption, resulting in mineralization defects and secondary hyperparathyroidism. Additionally, we demonstrate improved bone mineralization in patients with gastrectomy after gluconate therapy and showed the effectiveness of calcium gluconate over carbonate to balance impaired calcium hemostasis in mice. INTRODUCTION: Gastrectomy and hypochlorhydria due to long-term proton pump inhibitor therapy are associated with increased fracture risk because of intestinal calcium malabsorption. Hence, our objectives were to histologically investigate bone metabolism in patients with gastrectomy and to analyze the impact of calcium gluconate supplementation on skeletal integrity in the setting of impaired gastric acidification. METHODS: Undecalcified bone biopsies of 26 gastrectomized individuals were histologically analyzed. In the clinical setting, we retrospectively identified 5 gastrectomized patients with sufficient vitamin D level, who were additionally supplemented with calcium gluconate and had a real bone mineral density (aBMD) follow-up assessments. A mouse model of achlorhydria (ATP4b-/-) was used to compare the effect of calcium gluconate and calcium carbonate supplementation on bone metabolism. RESULTS: Biopsies from gastrectomized individuals showed significantly increased osteoid, osteoclast, and osteoblast indices and fibroosteoclasia (p < 0.05) as well as impaired calcium distribution in mineralized bone matrix compared to healthy controls. Five gastrectomized patients with sufficient vitamin D level demonstrated a significant increase in aBMD after a treatment with calcium gluconate alone for at least 6 months (p < 0.05). Calcium gluconate was superior to calcium carbonate in maintaining calcium metabolism in a mouse model of achlorhydria. CONCLUSION: Gastrectomy is associated with severe osteomalacia, marrow fibrosis, and impaired calcium distribution within the mineralized matrix. We show that calcium gluconate supplementation can increase bone mineral density in gastrectomized individuals and performs superior to calcium carbonate in restoring calcium/skeletal homoeostasis in a mouse model of achlorhydria.

Effects of extracellular phosphate on gene expression in murine osteoblasts.

That phosphate homeostasis is tightly linked to skeletal mineralization is probably best underscored by the fact that the phosphaturic hormone FGF23 is primarily expressed by terminally differentiated osteoblasts/osteocytes and that increased circulating FGF23 levels are causative for different types of hypophosphatemic rickets. In contrast, FGF23 inactivation results in hyperphosphatemia, and unexpectedly this phenotype is associated with severe osteomalacia in Fgf23-deficient mice. In this context it is interesting that different cell types have been shown to respond to extracellular phosphate, thereby raising the concept that phosphate can act as a signaling molecule. To identify phosphate-responsive genes in primary murine osteoblasts we performed genome wide expression analysis with cells maintained in medium containing either 1 or 4 mM sodium phosphate for 6 h. As confirmed by qRT-PCR, this analysis revealed that several known osteoblast differentiation markers (Bglap, Ibsp, and Phex) were unaffected by raising extracellular phosphate levels. In contrast, we found that the expression of Enpp1 and Ank, two genes encoding inhibitors of matrix mineralization, was induced by extracellular phosphate, while the expression of Sost and Dkk1, two genes encoding inhibitors of bone formation, was negatively regulated. The ability of osteoblasts to respond to extracellular phosphate was dependent on their differentiation state, and shRNA-dependent repression of the phosphate transporter Slc20a1 in MC3T3-E1 cells partially abolished their molecular response to phosphate. Taken together, our results provide further evidence for a role of extracellular phosphate as a signaling molecule and raise the possibility that severe hyperphosphatemia can negatively affect skeletal mineralization.

The role of the gastrointestinal tract in calcium homeostasis and bone remodeling.

While skeletal biology was approached in a rather isolated fashion in the past, an increasing understanding of the interplay between extraskeletal organs and bone remodeling has been obtained in recent years. This review will discuss recent advances in the field that have shed light on how the gastrointestinal tract and bone relate to each other. In particular, the importance of the GI tract in maintaining calcium homeostasis and skeletal integrity will be reviewed as impaired gastric acid production represents a major public health problem with possible implications for sufficient calcium absorption. Osteoporosis, the most prevalent bone disease worldwide, is caused not only by intrinsic defects affecting bone cell differentiation and function but also by a large set of extrinsic factors including hormonal disturbances, malnutrition, and iatrogenic drug application. Given the skeletal requirements of calcium, amino acids, and energy for bone turnover and renewal, it is not surprising that the gastrointestinal (GI) tract is of major importance for skeletal integrity.

Increased osteoblast and osteoclast indices in individuals with systemic mastocytosis.

UNLABELLED: Indolent systemic mastocytosis (ISM) can trigger bone loss. However, the clinical relevance of different mast cell infiltration patterns for bone remains to be clarified. Here, we report increased bone turnover in individuals with ISM, and its extent is rather related to the type of mast cell distribution within the bone marrow than to the presence or absence of cutaneous manifestations. INTRODUCTION: It is well established that ISM can trigger osteopenia or osteoporosis. However, neither the clinical relevance of the infiltration pattern of mast cells within the bone marrow nor the impact of the presence or absence of cutaneous mast cell infiltration has been elucidated. METHODS: We retrospectively analysed 300 cases with histologically proven ISM of the bone marrow and performed quantitative histomorphometry for a subgroup of 159 patients that did not receive any treatment before the biopsies were taken. Most importantly, since 66 % of the patients displayed ISM without the characteristic skin lesions, we were able to compare ISM with or without cutaneous manifestation. RESULTS: We found that both forms of ISM were not only characterized by a decreased trabecular bone mass but also by an increased number of osteoclasts and osteoblasts. Interestingly, when we analysed these data in relation to mast cell distribution, we found that the bone cell numbers in cases with mast cell granulomas were significantly increased compared to cases with diffuse mast cell distribution. Moreover, evidence of increased bone turnover was also found in 16 patients displaying osteosclerosis. CONCLUSION: Based on the largest cohort of bone biopsies from patients with ISM analysed so far, we could demonstrate high bone turnover, more specifically increased osteoblast and osteoclast numbers and surface indices, as a cause of the skeletal changes. Moreover, the severity of the bone disease is presumably rather dependent on the amount of mast cells and their distribution within the bone marrow irrespective of the presence or absence of cutaneous involvement.

Impaired bone mineralization accompanied by low vitamin D and secondary hyperparathyroidism in patients with femoral neck fracture.

SUMMARY: Although it is well established that a decrease in bone mass increases the risk of osteoporotic fractures, the proportion of fractures attributable to areal bone mineral density (BMD) is rather low. Here, we have identified bone mineralization defects together with low serum 25-hydroxyvitamin D (25-(OH) D) levels as additional factors associated with femoral neck fractures. INTRODUCTION: Osteoporotic fractures of the femoral neck are associated with increased morbidity and mortality. Although it is well established that a decrease in bone mass increases the risk of osteoporotic fractures, the proportion of fractures attributable to areal BMD is rather low. To identify possible additional factors influencing femur neck fragility, we analyzed patients with femoral neck fracture. METHODS: We performed a detailed clinical and histomorphometrical evaluation on 103 patients with femoral neck fracture including dual-energy X-ray absorptiometry, laboratory parameters, and histomorphometric and bone mineral density distribution (BMDD) analyses of undecalcified processed biopsies of the femoral head and set them in direct comparison to skeletal healthy control individuals. RESULTS: Patients with femoral neck fracture displayed significantly lower serum 25-(OH) D levels and increased serum parathyroid hormone (PTH) compared to controls. Histomorphometric analysis revealed not only a decreased bone volume and trabecular thickness in the biopsies of the patients, but also a significant increase of osteoid indices. BMDD analysis showed increased heterogeneity of mineralization in patients with femoral neck fracture. Moreover, patients with femoral neck fracture and serum 25-(OH) D levels below 12 µg/l displayed significantly thinner trabecular bone. CONCLUSION: Taken together, our data suggest that impaired bone mineralization accompanied by low serum 25-(OH) D levels is of major importance in the etiology of femoral neck fractures. Therefore, balancing serum 25-(OH) D levels and thereby normalizing PTH serum levels may counteract pronounced mineralization defects and might decrease the incidence of femoral neck fractures.

Mice over-expressing salmon calcitonin have strongly attenuated osteoarthritic histopathological changes after destabilization of the medial meniscus.

OBJECTIVE: Calcitonin is well-known for its inhibitory actions on bone-resorbing osteoclasts and recently potential beneficial effects on cartilage were shown. We investigated effects of salmon calcitonin (sCT) on the articular cartilage and bone, after destabilization of the medial meniscus (DMM) in normal and sCT over-expressing mice. DESIGN: Bone phenotype of transgenic (TG) C57Bl/6 mice over-expressing sCT at 6 months and 12 months was investigated by (1) serum osteocalcin and urinary deoxypyridinoline and (2) dynamic and normal histomorphometry of vertebrae bodies. In subsequent evaluation of cartilage and subchondral bone changes, 44 10-week old TG or wild-type (WT) mice were randomized into four groups and subjected to DMM or sham-operations. After 7 weeks animals were sacrificed, and knee joints were isolated for histological analysis. RESULTS: Trabecular bone volume (BV/TV) increased 150% after 6 months and 300% after 12 months in sCT-expressing mice when compared to WT controls (P<0.05). Osteoblast number, bone formation rate and osteocalcin measurements were not affected in TG mice over-expressing sCT. In WT animals, a 5-fold increase in the quantitative erosion index was observed after DMM, and the semi-quantitative OARSI score showed over 400% (P<0.001) increase, compared to sham-operated WT mice. DMM-operated TG mice were protected against cartilage erosion and showed a 65% and 64% (P<0.001) reduction, respectively, for the two histopathological evaluation methods. CONCLUSIONS: sCT over-expressing mice had higher bone volume, and were protected against cartilage erosion. These data suggest that increased levels of sCT may hamper the pathogenesis of osteoarthritis (OA). However more studies are necessary to confirm these preliminary results.

Skeletal mineralization defects in adult hypophosphatasia--a clinical and histological analysis.

UNLABELLED: Histomorphometry and quantitative backscattered electron microscopy of iliac crest biopsies from patients with adult hypophosphatasia not only confirmed the expected enrichment of non-mineralized osteoid, but also demonstrated an altered trabecular microarchitecture, an increased number of osteoblasts, and an impaired calcium distribution within the mineralized bone matrix. INTRODUCTION: Adult hypophosphatasia is an inherited disorder of bone metabolism caused by inactivating mutations of the ALPL gene, encoding tissue non-specific alkaline phosphatase. While it is commonly accepted that the increased fracture risk of the patients is the consequence of osteomalacia, there are only few studies describing a complete histomorphometric analysis of bone biopsies from affected individuals. Therefore, we analyzed iliac crest biopsies from eight patients and set them in direct comparison to biopsies from healthy donors or from individuals with other types of osteomalacia. METHODS: Histomorphometric analysis was performed on non-decalcified sections stained either after von Kossa/van Gieson or with toluidine blue. Bone mineral density distribution was quantified by backscattered electron microscopy. RESULTS: Besides the well-documented enrichment of non-mineralized bone matrix in individuals suffering from adult hypophosphatasia, our histomorphometric analysis revealed alterations of the trabecular microarchitecture and an increased number of osteoblasts compared to healthy controls or to individuals with other types of osteomalacia. Moreover, the analysis of the mineralized bone matrix revealed significantly decreased calcium content in patients with adult hypophosphatasia. CONCLUSIONS: Taken together, our data show that adult hypophosphatasia does not solely result in an enrichment of osteoid, but also in a considerable degradation of bone quality, which might contribute to the increased fracture risk of the affected individuals.

Wnt1 Promotes Cementum and Alveolar Bone Growth in a Time-Dependent Manner.

The WNT/ß-catenin signaling pathway plays a central role in the biology of the periodontium, yet the function of specific extracellular WNT ligands remains poorly understood. By using a Wnt1-inducible transgenic mouse model targeting Col1a1-expressing alveolar osteoblasts, odontoblasts, and cementoblasts, we demonstrate that the WNT ligand WNT1 is a strong promoter of cementum and alveolar bone formation in vivo. We induced Wnt1 expression for 1, 3, or 9 wk in Wnt1Tg mice and analyzed them at the age of 6 wk and 12 wk. Micro-computed tomography (CT) analyses of the mandibles revealed a 1.8-fold increased bone volume after 1 and 3 wk of Wnt1 expression and a 3-fold increased bone volume after 9 wk of Wnt1 expression compared to controls. In addition, the alveolar ridges were higher in Wnt1Tg mice as compared to controls. Nondecalcified histology demonstrated increased acellular cementum thickness and cellular cementum volume after 3 and 9 wk of Wnt1 expression. However, 9 wk of Wnt1 expression was also associated with periodontal breakdown and ectopic mineralization of the pulp. The composition of this ectopic matrix was comparable to those of cellular cementum as demonstrated by quantitative backscattered electron imaging and immunohistochemistry for noncollagenous proteins. Our analyses of 52-wk-old mice after 9 wk of Wnt1 expression revealed that Wnt1 expression affects mandibular bone and growing incisors but not molar teeth, indicating that Wnt1 influences only growing tissues. To further investigate the effect of Wnt1 on cementoblasts, we stably transfected the cementoblast cell line (OCCM-30) with a vector expressing Wnt1-HA and performed proliferation as well as differentiation experiments. These experiments demonstrated that Wnt1 promotes proliferation but not differentiation of cementoblasts. Taken together, our findings identify, for the first time, Wnt1 as a critical regulator of alveolar bone and cementum formation, as well as provide important insights for harnessing the WNT signal pathway in regenerative dentistry.

Identification of molecular markers for articular cartilage.

OBJECTIVE: The aim of the current study was to identify molecular markers for articular cartilage (AC) that can be used as tools for the quality control of tissue engineered (TE) cartilage. DESIGN: A genome-wide expression analysis was performed using RNA isolated from articular and growth plate (GP) cartilage, both extracted from the knee joints of 6 weeks old minipigs. After confirming the specific expression for selected genes by RT-PCR, these were used as molecular markers for the quality control of TE cartilage. RESULTS: Albeit several known chondrocyte markers were expressed to a similar extent in articular and GP cartilage, our genome-wide expression analysis led us to identify genes being selectively expressed in either GP or articular chondrocytes. These findings led us to perform a RT-PCR expression analysis for the corresponding genes to demonstrate the absence of GP-specific markers in TE cartilage, while common or AC markers were expressed. CONCLUSIONS: Taken together, these results provide important novel insights into chondrocyte biology in general and AC in particular. In addition, it is reasonable to speculate, that some of the identified genes play distinct roles in the regulation of articular chondrocyte differentiation and/or function, thereby raising the possibility that they may serve as targets for non-operative therapies of osteoarthritis (OA).

High bone turnover and accumulation of osteoid in patients with neurofibromatosis 1.

UNLABELLED: Although it is known that neurofibromatosis 1 (NF1) patients suffer from vitamin D deficiency and display decreased bone mineral density (BMD), a systematic clinical and histomorphometrical analysis is absent. Our data demonstrate that NF1 patients display high bone turnover and accumulation of osteoid and that supplementation of vitamin D has a beneficial effect on their BMD. INTRODUCTION: Neurofibromatosis 1 results in a wide range of clinical manifestations, including decreased BMD. Although it has been reported that NF1 patients have decreased vitamin D serum levels, the manifestation of the disease at the bone tissue level has rarely been analyzed. METHODS: Thus, we performed a clinical evaluation of 14 NF1 patients in comparison to age- and sex-matched control individuals. The analysis included dual X-ray absorptiometry osteodensitometry, laboratory parameters, histomorphometric and quantitative backscattered electron imaging (qBEI) analyses of undecalcified bone biopsies. RESULTS: NF1 patients display significantly lower 25-(OH)-cholecalciferol serum levels and decreased BMD compared to control individuals. Histomorphometric analysis did not only reveal a reduced trabecular bone volume in biopsies from NF1 patients, but also a significantly increased osteoid volume and increased numbers of osteoblasts and osteoclasts. Moreover, qBEI analysis revealed a significant decrease of the calcium content in biopsies from NF1 patients. To address the question whether a normalization of calcium homeostasis improves BMD in NF1 patients, we treated four patients with cholecalciferol for 1 year, which resulted in a significant increase of BMD. CONCLUSION: Taken together, our data provide the first complete histomorphometric analysis from NF1 patients. Moreover, they suggest that low vitamin D levels significantly contribute to the skeletal defects associated with the disease.

Preproenkephalin (Penk) is expressed in differentiated osteoblasts, and its deletion in Hyp mice partially rescues their bone mineralization defect.

Although our understanding of the molecular mechanisms controlling osteoblast differentiation and function is steadily increasing, there are still many open questions, especially regarding the regulation of bone matrix mineralization. For instance, while there is hallmark evidence for the importance of the endopeptidase Phex, whose inactivation in Hyp mice or human patients causes X-linked hypophosphatemic rickets, it is still largely unknown how Phex controls bone mineralization since a physiological substrate for its endopeptidase activity has not been identified yet. Using a genome-wide expression analysis comparing primary calvarial osteoblasts, we have identified preproenkephalin (Penk) as a gene that is selectively expressed in mineralized cultures. Since a role of enkephalin in the regulation of bone remodeling has been suggested previously and since Leu-enkephalin is known to be cleaved by Phex, we analyzed whether Penk expression in osteoblasts is physiologically relevant. Through skeletal analysis of a Penk-deficient mouse model, we found that Penk expression is dispensable for bone development and remodeling since we could not detect any defect following nondecalcified bone histology and histomorphometry compared to wild-type littermates. When Penk was deleted in Phex-deficient Hyp mice, however, we observed a significant reduction of the osteoid enrichment at 24 weeks of age, whereas their disturbance of mineral homeostasis was not affected by the additional absence of the Penk gene. Taken together, our data provide the first in vivo analysis concerning the role of Penk in osteoblasts.

The osteoblast: a sophisticated fibroblast under central surveillance.

The study of the biology of osteoblasts, or bone-forming cells, illustrates how mammalian genetics has profoundly modified our understanding of cell differentiation and physiologic processes. Indeed, genetic-based studies over the past 5 years have revealed how osteoblast differentiation is controlled through growth and transcription factors. Likewise, the recent identification, using mutant mouse models, of a central component in the regulation of bone formation expands our understanding of the control of bone remodeling. This regulatory loop, which involves the hormone leptin, may help to explain the protective effect of obesity on bone mass in humans. In addition, it provides a novel physiologic concept that may shed light on the etiology of osteoporosis and help to identify new therapeutic targets.

The protein tyrosine phosphatase Rptpzeta is expressed in differentiated osteoblasts and affects bone formation in mice.

Tyrosine phosphorylation of intracellular substrates is one mechanism to regulate cellular proliferation and differentiation. Protein tyrosine phosphatases (PTPs) act by dephosphorylation of substrates and thereby counteract the activity of tyrosine kinases. Few PTPs have been suggested to play a role in bone remodeling, one of them being Rptpzeta, since it has been shown to be suppressed by pleiotrophin, a heparin-binding molecule affecting bone formation, when over-expressed in transgenic mice. In a genome-wide expression analysis approach we found that Ptprz1, the gene encoding Rptpzeta, is strongly induced upon terminal differentiation of murine primary calvarial osteoblasts. Using RT-PCR and Western Blotting we further demonstrated that differentiated osteoblasts, in contrast to neuronal cells, specifically express the short transmembrane isoform of Rptpzeta. To uncover a potential role of Rptpzeta in bone remodeling we next analyzed the skeletal phenotype of a Rptpzeta-deficient mouse model using non-decalcified histology and histomorphometry. Compared to wildtype littermates, the Rptpzeta-deficient mice display a decreased trabecular bone volume at the age of 50 weeks, caused by a reduced bone formation rate. Likewise, Rptpzeta-deficient calvarial osteoblasts analyzed ex vivo display decreased expression of osteoblast markers, indicating a cell-autonomous defect. This was confirmed by the finding that Rptpzeta-deficient osteoblasts had a diminished potential to form osteocyte-like cellular extensions on Matrigel-coated surfaces. Taken together, these data provide the first evidence for a physiological role of Rptpzeta in bone remodeling, and thus identify Rptpzeta as the first PTP regulating bone formation in vivo.