F. Timo Beil

Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects

Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunB Δ/Δ mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junB Δ/Δ osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16INK4a levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage–osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.

Impaired gastric acidification negatively affects calcium homeostasis and bone mass.

Activation of osteoclasts and their acidification-dependent resorption of bone is thought to maintain proper serum calcium levels. Here we show that osteoclast dysfunction alone does not generally affect calcium homeostasis. Indeed, mice deficient in Src, encoding a tyrosine kinase critical for osteoclast activity, show signs of osteopetrosis, but without hypocalcemia or defects in bone mineralization. Mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells, have the expected defects in gastric acidification but also secondary hyperparathyroidism and osteoporosis and modest hypocalcemia. These results suggest that alterations in calcium homeostasis can be driven by defects in gastric acidification, especially given that calcium gluconate supplementation fully rescues the phenotype of the Cckbr-mutant mice. Finally, mice deficient in Tcirg1, encoding a subunit of the vacuolar proton pump specifically expressed in both osteoclasts and parietal cells, show hypocalcemia and osteopetrorickets. Although neither Src- nor Cckbr-deficient mice have this latter phenotype, the combined deficiency of both genes results in osteopetrorickets. Thus, we find that osteopetrosis and osteopetrorickets are distinct phenotypes, depending on the site or sites of defective acidification (pages 610–612).

Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells

Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL‐1) and IL‐18, two cytokines of the IL‐1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL‐33, another IL‐1 family member, has not been addressed yet. Since we observed that the expression of IL‐33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL‐33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1‐deficient mice, which lack the IL‐33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL‐33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL‐33 completely abolished the generation of TRACP+ multinucleated osteoclasts, even in the presence of RANKL and macrophage colony‐stimulating factor (M‐CSF). Although our molecular studies revealed that IL‐33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL‐33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL‐33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL‐33 as an inhibitor of bone resorption.

Control of bone formation by the serpentine receptor Frizzled-9.

Fzd9, induced upon osteoblast differentiation, is required for bone matrix mineralization in primary osteoblasts.

Paget's Disease of Bone: Histologic Analysis of 754 Patients

Although Pagets disease of bone (PDB) is the second most common metabolic bone disease, to our knowledge, there is only one quantitative analysis on the histological and especially on the histomorphometric level. Therefore, the aim of this study was to analyze, on the basis of the Hamburg Bone Register, PBD in terms of incidence, skeletal distribution, malignant transformation, and histological and histomorphometric characteristics. Bone biopsies and patient files of 754 cases with histologically proven PDB were reviewed in a retrospective study. Quantitative static histomorphometry was performed on a representative subgroup of 247 biopsies derived from patients with manifestation of PDB at the iliac crest and compared with an age‐ and sex‐matched control group. The peak incidence of PDB was between 70 and 80 yr of age. The majority of monostotic skeletal manifestation was localized at the os ilium, followed by the spine and femur. Histomorphometric results showed a high bone turnover with a significant increase in bone resorption and bone formation indices leading to an increased bone volume. Paget sarcoma was diagnosed in 6 of 754 patients, indicating a malignant transformation in 0.8% of the affected patients. Taken together, our study characterizes PDB in Germany on the basis of one of the largest cohorts of patients with histologically proven PDB. Moreover, for the first time, a quantitative histomorphometric approach was taken for >200 cases, where we could show local high bone mass lesions as a result of an increase of both osteoclast and osteoblast indices.

Apolipoprotein E-dependent inverse regulation of vertebral bone and adipose tissue mass in C57Bl/6 mice: Modulation by diet-induced obesity

The long prevailing view that obesity is generally associated with beneficial effects on the skeleton has recently been challenged. Apolipoprotein E (apoE) is known to influence both adipose tissue and bone. The goal of the current study was to examine the impact of apoE on the development of fat mass and bone mass in mice under conditions of diet-induced obesity (DIO). Four week-old male C57BL/6 (WT) and apoE-deficient (apoE(-/-)) mice received a control or a diabetogenic high-fat diet (HFD) for 16 weeks. The control-fed apoE(-/-) animals displayed less total fat mass and higher lumbar trabecular bone volume (BV/TV) than WT controls. When stressed with HFD to induce obesity, apoE(-/-) mice had a lower body weight, lower serum glucose, insulin and leptin levels and accumulated less white adipose tissue mass at all sites including bone marrow. While WT animals showed no significant change in BV/TV and bone formation rate (BFR), apoE deficiency led to a decrease of BV/TV and BFR when stressed with HFD. Bone resorption parameters were not affected by HFD in either genotype. Taken together, under normal dietary conditions, apoE-deficient mice acquire less fat mass and more bone mass than WT littermates. When stressed with HFD to develop DIO, the difference of total body fat mass becomes larger and the difference of bone mass smaller between the genotypes. We conclude that apoE is involved in an inverse regulation of bone mass and fat mass in growing mice and that this effect is modulated by diet-induced obesity.

BMP-7-induced ectopic bone formation and fracture healing is impaired by systemic NSAID application in C57BL/6-mice.

Nonsteroidal antiinflammatory drugs (NSAIDs) are known to potentially impair the fracture healing process. The aim of the present study was to determine if the impairment of bone healing by systemic NSAID application is, at least in part, due to an interaction of NSAIDs with the bone anabolic BMP‐7 pathway. Therefore, we first analyzed fracture healing in control and diclofenac‐treated mice, where we not only found a significant impairment of fracture healing due to diclofenac treatment as assessed by biomechanical testing and µCT imaging, but also found high coexpression of bone morphogenetic protein‐7 (BMP‐7) and cyclooxygenase‐2 (COX‐2) within the fracture callus of both groups. To experimentally address the possible interaction between BMP‐7 and COX‐2, we then induced ectopic bone formation in control (n = 10) and diclofenac‐treated mice (n = 10) by application of BMP‐7 (recombinant human OP‐1, rhOP‐1) into the hamstring muscles. After 20 days of treatment, each ectopic bone nodule was analyzed by contact‐radiography, µCT, histology, and histomorphometry. Diclofenac application decreased the trabecular number and bone mass in the ectopic bone nodules significantly due to reduced osteoblast number and activity. These data demonstrate that the bone anabolic effect of BMP‐7 and fracture healing is impaired by diclofenac application, and suggest that the potential negative impact of NSAIDs on fracture healing is, at least in part, due to interference with BMP‐7 signaling.

Skeletal mineralization defects in adult hypophosphatasia—a clinical and histological analysis

SummaryHistomorphometry 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.IntroductionAdult 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.MethodsHistomorphometric 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.ResultsBesides 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.ConclusionsTaken 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.

68Ga DOTA-TATE PET/CT allows tumor localization in patients with tumor-induced osteomalacia but negative 111In-octreotide SPECT/CT

Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome characterized by renal phosphate wasting, hypophosphatemia and low calcitriol levels as well as clinical symptoms like diffuse bone and muscle pain, fatigue fractures or increased fracture risk. Conventional imaging methods, however, often fail to detect the small tumors. Lately, tumor localization clearly improved by somatostatin-receptor (SSTR) imaging, such as octreotide scintigraphy or octreotide SPECT/CT. However, recent studies revealed that still a large number of tumors remained undetected by octreotide imaging. Hence, studies focused on different SSTR imaging methods such as 68Ga DOTA-NOC, 68Ga DOTA-TOC and 68Ga DOTA-TATE PET/CT with promising first results. Studies comparing different SSTR imaging methods for tumor localization in TIO are rare and thus little is known about diagnostic alternatives once a particular method failed to detect a tumor in patients with TIO. Here, we report the data of 5 consecutive patients suffering from TIO, who underwent both 111Indium-octreotide scintigraphy (111In-OCT) SPECT/CT as well as 68Ga DOTA-TATE PET/CT for tumor detection. While 111In-OCT SPECT/CT allowed tumor detection in only 1 of 5 patients, 68Ga DOTA-TATE PET/CT was able to localize the tumor in all patients. Afterwards, anatomical imaging of the region of interest was performed with CT and MRI. Thus, successful surgical resection of the tumor was achieved in all patients. Serum phosphate levels returned to normal and all patients reported relief of symptoms within weeks. Moreover, an iliac crest biopsy was obtained from every patient and revealed marked osteomalacia in all cases. Follow-up DXA revealed an increase in BMD of up to 34.5% 1-year postoperative, indicating remineralization. No recurrence was observed. In conclusion our data indicates that 68Ga DOTA-TATE PET/CT is an effective and promising diagnostic tool in the diagnosis of TIO, even in patients in whom 111In-OCT prior failed to detect a tumor.

Impaired bone formation and increased osteoclastogenesis in mice lacking chemokine (C-C motif) ligand 5 (Ccl5)

Chemokines play crucial roles in the recruitment of specific hematopoietic cell types, and some of them have been suggested to be involved in the regulation of bone remodeling. Because we have previously observed that chemokine (C‐C motif) ligand 2 (Ccl2) and Ccl5 are direct target genes of noncanonical Wnt signaling in osteoblasts, we analyzed the skeletal phenotypes of Ccl2‐deficient and Ccl5‐deficient mice. In line with previous studies, Ccl2‐deficient mice display a moderate reduction of osteoclastogenesis at the age of 6 months. In contrast, 6‐month‐old Ccl5‐deficient mice display osteopenia associated with decreased bone formation and increased osteoclastogenesis. Moreover, unlike in wild‐type and Ccl2‐deficient mice, large areas of their trabecular and endocortical bone surfaces are not covered by osteoblasts or bone‐lining cells, and this is associated with a severe reduction of endosteal bone formation. Although this phenotype diminishes with age, it is important that we could further identify a reduced number of osteal macrophages in 6‐month‐old Ccl5‐deficient mice, because this cell type has previously been reported to promote endosteal bone formation. Because Ccl5‐deficient mice also display increased osteoclastogenesis, we finally addressed the question of whether osteal macrophages could differentiate into osteoclasts and/or secrete inhibitors of osteoclastogenesis. For that purpose we isolated these cells by CD11b affinity purification from calvarial cultures and characterized them ex vivo. Here we found that they are unable to differentiate into osteoblasts or osteoclasts, but that their conditioned medium mediates an antiosteoclastogenic effect, possibly caused by interleukin‐18 (IL‐18), an inhibitor of osteoclastogenesis expressed by osteal macrophages. Taken together, our data provide in vivo evidence supporting the previously suggested role of Ccl5 in bone remodeling. Moreover, to the best of our knowledge, Ccl5‐deficient mice represent the first model with a spontaneous partial deficiency of osteal macrophages, a recently identified cell type, whose impact on bone remodeling is just beginning to be understood.