Anke Baranowsky

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).

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

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

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.

Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2.

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.

Gap junctional communication in human osteoclasts in vitro and in vivo

Bone‐forming cells are known to be coupled by gap junctions, formed primarily by connexin43 (Cx43). The role of Cx43 in osteoclasts has so far only been studied in rodents, where Cx43 is important for fusion of mononuclear precursors to osteoclasts. Given the potential importance for human diseases with pathologically altered osteoclasts, we asked whether a similar influence of Cx43 can also be observed in osteoclasts of human origin. For this purpose, Cx43 mRNA expression was studied in a time course experiment of human osteoclast differentiation by RT‐PCR. Localization of Cx43 in these cells was determined by immunohistochemistry and confocal microscopy. For the assessment of the effect of gap junction inhibition on cell fusion, gap junctions were blocked with heptanol during differentiation of the cells and the cells were then evaluated for multinuclearity. Paraffin sections of healthy bone and bone from patients with Pagets disease and giant cell tumour of the bone were used to study Cx43 expression in vivo. We found mRNA and protein expression of Cx43 in fully differentiated osteoclasts as well as in precursor cells. This expression decreased in the course of differentiation. Consistently, we found a lower expression of Cx43 in osteoclasts than in bone marrow precursor cells in the histology of healthy human bone. Blockade of gap junctional communication by heptanol led to a dose‐dependent decrease in multinuclearity, suggesting that gap junctional communication precedes cell fusion of human osteoclasts. Indeed, we found a particularly strong expression of Cx43 in the giant osteoclasts of patients with Pagets disease and giant cell tumour of the bone. These results show that gap junctional communication is important for fusion of human mononuclear precursor cells to osteoclasts and that gap junctional Cx43 might play a role in the regulation of size and multinuclearity of human osteoclasts in vivo.

Increased trabecular bone formation in mice lacking the growth factor midkine

Midkine (Mdk) and pleiotrophin (Ptn) comprise a family of heparin‐binding growth factors known primarily for their effects on neuronal cells. Since transgenic mice overexpressing Ptn have been reported to display increased bone density, we have previously analyzed Ptn‐deficient mice but failed to detect any abnormality of skeletal development and remodeling. Together with the finding that Mdk expression increases in the course of primary osteoblast differentiation, we reasoned that Mdk, rather than Ptn, could play a physiologic role in bone formation. Here, we show that Mdk‐deficient mice display an increased trabecular bone volume at 12 and 18 months of age, accompanied by cortical porosity. Histomorphometric quantification demonstrated an increased bone‐formation rate compared with wild‐type littermates, whereas bone resorption was differentially affected in trabecular and cortical bone of Mdk‐deficient mice. To understand the effect of Mdk on bone formation at the molecular level, we performed a genome‐wide expression analysis of primary osteoblasts and identified Ank and Enpp1 as Mdk‐induced genes whose decreased expression in Mdk‐deficient osteoblasts may explain, at least in part, the observed skeletal phenotype. Finally, we performed ovariectomy and observed bone loss only in wild‐type but not in Mdk‐deficient animals. Taken together, our data demonstrate that Mdk deficiency, at least in mice, results in an increased trabecular bone formation, thereby raising the possibility that Mdk‐specific antagonists might prove beneficial in osteoporosis therapy.

Osteolytic prostate cancer cells induce the expression of specific cytokines in bone-forming osteoblasts through a Stat3/5-dependent mechanism

Prostate cancer primarily metastasizes to bone, and the interaction of cancer cells with bone cells results in a local activation of bone formation and/or bone resorption. Since the cellular and molecular mechanisms underlying the development of these tumor-induced osteoblastic or osteolytic lesions are still poorly understood, we have compared the effects of two prostate cancer cell lines, osteoblastic MDA-PCa-2b cells and osteolytic PC-3 cells, on bone-forming osteoblasts. Using Affymetrix Gene Chip hybridization followed by qRT-PCR confirmation we were able to identify specific genes, including Smpd3 and Dmp1, whose expression is significantly reduced upon treatment with PC-3-conditioned medium. Moreover, we observed that PC-3-conditioned medium led to a marked induction of several cytokine genes, including Cxcl5, Cxcl12 and Tnfsf11, the latter one encoding for the osteoclast differentiation factor Rankl. Likewise, when we analyzed the effects of MDA-PCa-2b- and PC-3-conditioned medium on signal transduction in osteoblasts we did not only observe opposite effects on the canonical Wnt signalling pathway, but also a specific induction of Erk and Stat phosphorylation by PC-3-conditioned medium. Most importantly, the induction of Cxcl5, Cxcl12 and Tnfsf11 in osteoblasts by PC-3-conditioned medium was abrogated by the Stat3/5 inhibitor piceatannol, whereas the selective blockade of Stat1 and Erk activation had no effect. Together with the finding, that activated Stat3 in osteoblasts was detectable in bone biopsies from patients with osteolytic metastases, our data suggest that the Stat3/5-dependent activation of cytokine expression in osteoblasts may have a significant impact on cancer cell migration and proliferation, but also on osteoclast activation.

p65-Dependent production of interleukin-1β by osteolytic prostate cancer cells causes an induction of chemokine expression in osteoblasts

Skeletal metastases are a frequent complication of prostate, breast and lung cancer, and the interactions of tumor cells with bone-forming osteoblasts and bone-resorbing osteoclasts have been suggested to play critical roles in disease progression. We have previously shown that treatment of primary murine osteoblasts with conditioned medium of the human osteolytic prostate cancer cell line PC-3 results in a rapid induction of chemokine expression, thereby providing further evidence for a molecular crosstalk between bone and tumor cells. The aim of our current study was to identify PC-3-derived molecules mediating this effect. Using Affymetrix Gene Chip hybridization followed by qRT-PCR we were able to confirm that the expression of chemokine-encoding genes is markedly induced in human primary osteoblasts following incubation with PC-3-conditioned medium. Since this induction was significantly affected upon alteration of p65-levels in PC-3 cells, we performed a second genome-wide expression analysis to identify p65-regulated cytokines, which were then tested for their ability to induce chemokine expression. Here we observed that interleukin-1β (IL-1B) did not only increase the expression of chemokines in osteoblasts, but also the phosphorylation of p65 and thereby its own expression. Since immunohistochemistry on bone biopsy sections from prostate cancer metastases demonstrated IL-1B expression in both, tumor cells and osteoblasts, our data suggest that IL-1B is one of the relevant cytokines involved in the skeletal complications of cancer metastases.

Increased Expression of Transthyretin in Leptin-Deficient ob/ob Mice is not Causative for Their Major Phenotypic Abnormalities

The hormone leptin is a critical regulator of adipogenesis and energy metabolism. Similarly, leptin‐deficient ob/ob mice display various metabolic abnormalities, including not only obesity and insulin resistance, but also hypogonadism and high bone mass. By genome‐wide expression analysis using hypothalamus RNA from wild‐type and ob/ob mice, we observed the increased expression of the gene for transthyretin (Ttr) in the latter, as confirmed by quantitative real‐time‐polymerase chain reaction. Because Ttr encodes a carrier protein for retinol transport, and because we further found increased retinol levels in the serum of ob/ob mice, we investigated whether the additional absence of Ttr would influence the ob/ob phenotype. It was found that Ttr‐deficient ob/ob mice were indistinguishable from ob/ob littermates in terms of body weight, as well as serum glucose, insulin and cholesterol levels. Although all of these parameters were identical to wild‐type controls in Ttr‐deficient mice, we found that the sole deletion of Ttr caused a significant increase of trabecular bone mass, bone marrow adiposity and mean adipocyte area in white adipose tissue. Interestingly, all these latter parameters were highest in Ttr‐deficient ob/ob mice, and only in these mice did we observe a full penetrance of liver steatosis at 24 weeks of age. Taken together, our data demonstrate that the increased expression of Ttr in ob/ob mice does not cause (but rather attenuates) their phenotypic abnormalities.