Michaela Kneissel

Osteocyte Wnt/β-Catenin Signaling Is Required for Normal Bone Homeostasis

ABSTRACT β-Catenin-dependent canonical Wnt signaling plays an important role in bone metabolism by controlling differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. To investigate its function in osteocytes, the cell type constituting the majority of bone cells, we generated osteocyte-specific β-catenin-deficient mice (Ctnnb1loxP/loxP; Dmp1-Cre). Homozygous mutants were born at normal Mendelian frequency with no obvious morphological abnormalities or detectable differences in size or body weight, but bone mass accrual was strongly impaired due to early-onset, progressive bone loss in the appendicular and axial skeleton with mild growth retardation and premature lethality. Cancellous bone mass was almost completely absent, and cortical bone thickness was dramatically reduced. The low-bone-mass phenotype was associated with increased osteoclast number and activity, whereas osteoblast function and osteocyte density were normal. Cortical bone Wnt/β-catenin target gene expression was reduced, and of the known regulators of osteoclast differentiation, osteoprotegerin (OPG) expression was significantly downregulated in osteocyte bone fractions of mutant mice. Moreover, the OPG levels expressed by osteocytes were higher than or comparable to the levels expressed by osteoblasts during skeletal growth and at maturity, suggesting that the reduction in osteocytic OPG and the concomitant increase in osteocytic RANKL/OPG ratio contribute to the increased number of osteoclasts and resorption in osteocyte-specific β-catenin mutants. Together, these results reveal a crucial novel function for osteocyte β-catenin signaling in controlling bone homeostasis.

Mice Lacking Histone Deacetylase 6 Have Hyperacetylated Tubulin but Are Viable and Develop Normally

ABSTRACT Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, α-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.

High Bone Resorption in Adult Aging Transgenic Mice Overexpressing Cbfa1/Runx2 in Cells of the Osteoblastic Lineage

ABSTRACT The runt family transcription factor core-binding factor α1 (Cbfa1) is essential for bone formation during development. Surprisingly, transgenic mice overexpressing Cbfa1 under the control of the 2.3-kb collagen type I promoter developed severe osteopenia that increased progressively with age and presented multiple fractures. Analysis of skeletally mature transgenic mice showed that osteoblast maturation was affected and that specifically in cortical bone, bone resorption as well as bone formation was increased, inducing high bone turnover rates and a decreased degree of mineralization. To understand the origin of the increased bone resorption, we developed bone marrow stromal cell cultures and reciprocal coculture of primary osteoblasts and spleen cells from wild-type or transgenic mice. We showed that transgenic cells of the osteoblastic lineage induced an increased number of tartrate-resistant acid phosphatase-positive multinucleated cells, suggesting that primary osteoblasts as well as bone marrow stromal cells from transgenic mice have stronger osteoclastogenic properties than cells derived from wild-type animals. We investigated the candidate genes whose altered expression could trigger this increase in bone resorption, and we found that the expression of receptor activator of NF-κB ligand (RANKL) and collagenase 3, two factors involved in bone formation-resorption coupling, was markedly increased in transgenic cells. Our data thus suggest that overexpression of Cbfa1 in cells of the osteoblastic lineage does not necessarily induce a substantial increase in bone formation in the adult skeleton but has a positive effect on osteoclast differentiation in vitro and can also dramatically enhance bone resorption in vivo, possibly through increased RANKL expression.

Control of the SOST Bone Enhancer by PTH Using MEF2 Transcription Factors

Expression of the osteocyte‐derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH.

Parathyroid Hormone (PTH)–Induced Bone Gain Is Blunted in SOST Overexpressing and Deficient Mice

Intermittent parathyroid hormone (PTH) treatment is a potent bone anabolic principle that suppresses expression of the bone formation inhibitor Sost. We addressed the relevance of Sost suppression for PTH‐induced bone anabolism in vivo using mice with altered Sost gene dosage. Six‐month‐old Sost overexpressing and 2‐month‐old Sost deficient male mice and their wild‐type littermates were subjected to daily injections of 100 µg/kg PTH(1–34) or vehicle for a 2‐month period. A follow‐up study was performed in Sost deficient mice using 40 and 80 µg/kg PTH(1–34). Animals were sacrificed 4 hours after the final PTH administration and Sost expression in long bone diaphyses was determined by qPCR. Bone changes were analyzed in vivo in the distal femur metaphysis by pQCT and ex vivo in the tibia and lumbar spine by DXA. Detailed ex vivo analyses of the femur were performed by pQCT, µCT, and histomorphometry. Overexpression of Sost resulted in osteopenia and Sost deletion in high bone mass. As shown before, PTH suppressed Sost in wild‐type mice. PTH treatment induced substantial increases in bone mineral density, content, and cortical thickness and in aging wild‐type mice also led to cancellous bone gain owing to amplified bone formation rates. PTH‐induced bone gain was blunted at all doses and skeletal sites in Sost overexpressing and deficient mice owing to attenuated bone formation rates, whereas bone resorption was not different from that in PTH‐treated wild‐type controls. These data suggest that suppression of the bone formation inhibitor Sost by intermittent PTH treatment contributes to PTH bone anabolism.

Does osteocytic SOST suppression mediate PTH bone anabolism

Parathyroid hormone (PTH) has bone anabolic activity when administered intermittently, affecting cells of the osteoblastic lineage at various stages, yet much remains to be learned about precisely how PTH promotes osteoblastic bone formation. Recent discoveries revealed that PTH causes transcriptional suppression of the osteocyte marker gene SOST, which encodes the potent secreted bone formation inhibitor, sclerostin. This review addresses whether osteocytes, terminally differentiated cells of the osteoblastic lineage, which are entrapped within the mineralized bone matrix, contribute to PTH-induced bone formation responses via regulation of sclerostin levels, and discusses recent evidence on how the bone anabolic responses elicited by intermittent PTH treatment or by sclerostin inhibition overlap and diverge.

Bone Tissue and Its Mineralization in Aged Estrogen-Depleted Rats After Long-term Intermittent Treatment With Parathyroid Hormone (PTH) Analog SDZ PTS 893 or Human PTH(1-34)

Intermittently administered parathyroid hormone (PTH) is a potent bone anabolic agent. We aimed to determine the impact of long-term treatment with PTH on bone structure, dynamics, and mineralization. We ovariectomized (ovx) 1-year-old rats with the exception of a baseline and a sham-operated group. Twelve weeks later, a 36 week treatment with PTH analog SDZ PTS 893 (12.5, 25, 50, 100 microg/kg), human PTH(1-34) (25, 50, 100 microg/kg), or vehicle (ovx, sham) was initiated. Bone dynamics, structure, and mineralization were evaluated in the lumbar spine and in the femoral diaphysis. Cancellous bone turnover was elevated 12 weeks postovariectomy in estrogen-deficient, vehicle-treated animals, but returned to the level of the sham group by 48 weeks. The animals experienced substantial cancellous bone loss associated with a reduction of trabecular number and presented with a partly rod-like trabecular network. After 36 weeks of treatment with SDZ PTS 893 or human PTH(1-34), cancellous bone formation rates and turnover were raised in all treated groups compared with age-matched controls. The mineral apposition rate was increasing with dose. This amplified matrix synthesis led to trabecular thickening, but not to an increase in trabecular number, resulting in a crude, plate-like cancellous network with a high bone volume fraction. Fluorochrome label-based cortical bone dynamics demonstrated that a thick ring of new bone was formed at the endocortex by activation of modeling drifts during treatment. Treatment-induced cortical bone formation was increased with dose at the subperiosteal and endocortical envelopes, but substantially higher at the latter. Intracortical bone turnover was elevated near the endocortex. Bone mineralization was undisturbed in all compartments. The average degree of mineralization was lowered slightly, reflecting the increased portion of new bone formed during treatment. In summary, the main anabolic effect was mediated for both peptides by an increase in bone apposition with dose, persisting throughout treatment that lasted more than one third of the lifespan of the rats, and direct activation of bone-forming surfaces. As a result, a substantial amount of new bone, maintained at elevated turnover and adequate mineralization levels, formed predominantly at compartments exposed to bone marrow.

Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23-mediated hypophosphatemic rickets

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25‐dihydroxyvitamin D3 (1,25[OH]2D3). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2D3 levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X‐linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23‐mediated hypophosphatemic disorders using NVP‐BGJ398, a novel selective, pan‐specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1‐null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long‐term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP‐BGJ398 treatment as a novel approach for the therapy of FGF23‐mediated hypophosphatemic diseases.

Age- and Sex-Dependent Cancellous Bone Changes in a 4000y BP Population

We studied cancellous bone loss in a 4000y BP population, using several methods designed to detect age-related changes, in order to investigate the pattern of cancellous bone loss in this ancient population and to compare the results deriving from different methods used on identical specimens. We used 10-mm sections of fourth lumbar vertebral bodies and left femoral necks of 18 individuals of both sexes with estimated ages from 20 to 60 years of a 4000y BP bronze-age population. Stereoscopic photographs were used for three-dimensional analysis and trabecular number (TN) counting. After embedding, the following parameters were measured in different image analysis systems using plane parallel block samples: bone mineral density (BMD) in water by DEXA, and by evaluation of standardized radiographic images; fractional bone volume (BV/TV) in backscattered electron images of the trabecular surface layer and in optical images of trabeculae in a surface-stained layer; and trabecular bone pattern factor (TBPf) in the latter images. There was a high correlation between the results of morphological methods for measuring fractional bone volume. Reasonable correlations were found between the x-ray photon methods and poor correlations between these and the morphological methods. These poor correlations may be due to the diagenetic substitution occurring in archaeological skeletons, which would strongly influence x-ray-based density measurements. However, all the methods demonstrated that the most dramatic loss of quantity and quality in cancellous bone occurred in females between 40 and 60 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Disruption of Lrp4 function by genetic deletion or pharmacological blockade increases bone mass and serum sclerostin levels

Significance Targeting WNT (Wingless-type)/β-catenin signaling has emerged as an attractive novel therapeutic approach to the treatment of bone diseases. We previously identified LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of action of the WNT signaling antagonist SOST/sclerostin in vitro. Here, we generated bone-specific Lrp4-deficient mouse lines and anti-LRP4 antibodies selectively disrupting the Lrp4 sclerostin facilitator function. Using these novel genetic and pharmacological tools, we demonstrate that disruption of Lrp4 function induces bone gain in vivo and results in highly elevated circulating sclerostin levels. Together, these findings provide important novel insights into the role of LRP4 as a key regulator of bone homeostasis and into the mode of action of sclerostin and provide a new strategy for promoting bone gain through targeting of the WNT pathway. We identified previously in vitro LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of the WNT (Wingless-type) antagonist sclerostin and found mutations disrupting this function to be associated with high bone mass in humans similar to patients lacking sclerostin. To further delineate the role of LRP4 in bone in vivo, we generated mice lacking Lrp4 in osteoblasts/osteocytes or osteocytes only. Lrp4 deficiency promoted progressive cancellous and cortical bone gain in both mutants, although more pronouncedly in mice deficient in osteoblast/osteocyte Lrp4, consistent with our observation in human bone that LRP4 is most strongly expressed by osteoblasts and early osteocytes. Bone gain was related primarily to increased bone formation. Interestingly, Lrp4 deficiency in bone dramatically elevated serum sclerostin levels whereas bone expression of Sost encoding for sclerostin was unaltered, indicating that osteoblastic Lrp4 retains sclerostin within bone. Moreover, we generated anti-LRP4 antibodies selectively blocking sclerostin facilitator function while leaving unperturbed LRP4–agrin interaction, which is essential for neuromuscular junction function. These antibodies increased bone formation and thus cancellous and cortical bone mass in skeletally mature rodents. Together, we demonstrate a pivotal role of LRP4 in bone homeostasis by retaining and facilitating sclerostin action locally and provide a novel avenue to bone anabolic therapy by antagonizing LRP4 sclerostin facilitator function.