Roland Baron

LDL Receptor-Related Protein 5 (LRP5) Affects Bone Accrual and Eye Development

In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.

WNT signaling in bone homeostasis and disease: from human mutations to treatments

Low bone mass and strength lead to fragility fractures, for example, in elderly individuals affected by osteoporosis or children with osteogenesis imperfecta. A decade ago, rare human mutations affecting bone negatively (osteoporosis-pseudoglioma syndrome) or positively (high–bone mass phenotype, sclerosteosis and Van Buchem disease) have been identified and found to all reside in components of the canonical WNT signaling machinery. Mouse genetics confirmed the importance of canonical Wnt signaling in the regulation of bone homeostasis, with activation of the pathway leading to increased, and inhibition leading to decreased, bone mass and strength. The importance of WNT signaling for bone has also been highlighted since then in the general population in numerous genome-wide association studies. The pathway is now the target for therapeutic intervention to restore bone strength in millions of patients at risk for fracture. This paper reviews our current understanding of the mechanisms by which WNT signalng regulates bone homeostasis.

BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop.

Wnt/β‐catenin signaling has recently been suggested to be involved in bone biology. The precise role of this cascade in osteoblast differentiation was examined. We show that a Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP‐2 in pre‐osteoblastic cells.

Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass

Wnt/β‐catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation.

Rescue of the Skeletal Phenotype of Vitamin D Receptor- Ablated Mice in the Setting of Normal Mineral Ion Homeostasis: Formal Histomorphometric and Biomechanical Analyses*

1,25-Dihydroxyvitamin D3 has been shown to play an important role in vitro in regulating osteoblast gene transcription and promoting osteoclast differentiation. To address the role of the vitamin D receptor (VDR) in skeletal homeostasis, formal histomorphometric analyses were performed in VDR null mice in the setting of impaired mineral ion homeostasis as well as in VDR null mice in whom normal mineral ion homeostasis had been preserved. In hypocalcemic VDR null mice, there was an increase in bone volume as a result of a dramatic increase in osteoid. There was also an increase in the number of osteoblasts without a significant change in the number of osteoclasts. Examination of the growth plate revealed marked disorganization, with an increase in vascularity and matrix. Biomechanical parameters demonstrated increased bone fragility in the hypocalcemic VDR null mice. In the VDR ablated mice in whom normal mineral ion homeostasis had been preserved, none of these measurements was significantly different fro...

Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

Spred is a Sprouty-related suppressor of Ras signalling.

Cellular proliferation, and differentiation of cells in response to extracellular signals, are controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Here we describe two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signalling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibited growth-factor-mediated activation of MAP kinase. The Ras–MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection revealed that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associated with Ras but did not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibited the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras–Raf interaction and MAP kinase signalling.

Activated parathyroid hormone/parathyroid hormone–related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone

Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. To assess the role of the PTH/PTH-related protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansens metaphyseal chondrodysplasia. In these transgenic mice, osteoblastic function was increased in the trabecular and endosteal compartments, whereas it was decreased in the periosteum. In trabecular bone of the transgenic mice, there was an increase in osteoblast precursors, as well as in mature osteoblasts. Osteoblastic expression of the constitutively active PPR induced a dramatic increase in osteoclast number in both trabecular and compact bone in transgenic animals. The net effect of these actions was a substantial increase in trabecular bone volume and a decrease in cortical bone thickness of the long bones. These findings, for the first time to our knowledge, identify the PPR as a crucial mediator of both bone-forming and bone-resorbing actions of PTH, and they underline the complexity and heterogeneity of the osteoblast population and/or their regulatory microenvironment.

Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-β in bone remodeling in females but not in males

To determine the contributions of estrogen receptor (ER)alpha and ERbeta in bone growth and remodeling in male and female mice, we generated and analyzed full knockouts for each receptor, and a double ER knockout. Although suppression of the ligand to the ERs (i.e., estradiol) after menopause or gonadectomy in females led to a catastrophic increase in bone turnover and concomitant bone loss, deletion of one or both ERs failed to show such an effect. Complete deletion of ERalpha led to a decrease, not an increase, in bone turnover and an increase, not a decrease, in trabecular bone volume in both male and female animals. Deletion of ERbeta led to different responses in males, where bone was unaffected, and in females, where bone resorption was decreased and trabecular bone volume increased. In contrast, deletion of both ERs led to a profound decrease in trabecular bone volume in females, which was associated with a decrease, not an increase, in bone turnover. Finally, deletion of ERalpha, but not ERbeta, led to major changes in circulating levels of estradiol and/or testosterone, indirectly affecting bone remodeling and bone mass. Thus, only ERalpha was shown to regulate bone remodeling in males, whereas in females both receptor subtypes influenced this process and could, at least under basal knockout conditions, compensate for each other.

Overexpression of ΔFosB transcription factor(s) increases boneformation and inhibits adipogenesis

Members of the AP-1 family of transcription factors participate in the regulation of bone cell proliferation and differentiation. We report here a potent AP-1-related regulator of osteoblast function: ΔFosB, a naturally occurring truncated form of FosB that arises from alternative splicing of the fosB transcript and is expressed in osteoblasts. Overexpression of ΔFosB in transgenic mice leads to increased bone formation throughout the skeleton and a continuous post-developmental increase in bone mass, leading to osteosclerosis. In contrast, ΔFosB inhibits adipogenesis both in vivo and in vitro, and downregulates the expression of early markers of adipocyte differentiation. Because osteoblasts and adipocytes are thought to share a common precursor, it is concluded that ΔFosB transcriptionally regulates osteoblastogenesis, possibly at the expense of adipogenesis.