Riccardo Chiusaroli

Potential Role for ADAM15 in Pathological Neovascularization in Mice

ABSTRACT ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15−/− mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15−/− mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15−/− mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15−/− mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.

Essential Role for ADAM19 in Cardiovascular Morphogenesis

ABSTRACT Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19−/− animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.

ΔFosB Induces Osteosclerosis and Decreases Adipogenesis by Two Independent Cell-Autonomous Mechanisms

ABSTRACT Osteoblasts and adipocytes may develop from common bone marrow mesenchymal precursors. Transgenic mice overexpressing ΔFosB, an AP-1 transcription factor, under the control of the neuron-specific enolase (NSE) promoter show both markedly increased bone formation and decreased adipogenesis. To determine whether the two phenotypes were linked, we targeted overexpression of ΔFosB in mice to the osteoblast by using the osteocalcin (OG2) promoter. OG2-ΔFosB mice demonstrated increased osteoblast numbers and an osteosclerotic phenotype but normal adipocyte differentiation. This result firmly establishes that the skeletal phenotype is cell autonomous to the osteoblast lineage and independent of adipocyte formation. It also strongly suggests that the decreased fat phenotype of NSE-ΔFosB mice is independent of the changes in the osteoblast lineage. In vitro, overexpression of ΔFosB in the preadipocytic 3T3-L1 cell line had little effect on adipocyte differentiation, whereas it prevented the induction of adipogenic transcription factors in the multipotential stromal cell line ST2. Also, ΔFosB isoforms bound to and altered the DNA-binding capacity of C/EBPβ. Thus, the inhibitory effect of ΔFosB on adipocyte differentiation appears to occur at early stages of stem cell commitment, affecting C/EBPβ functions. It is concluded that the changes in osteoblast and adipocyte differentiation in ΔFosB transgenic mice result from independent cell-autonomous mechanisms.

Deletion of the gene encoding c-Cbl alters the ability of osteoclasts to migrate, delaying resorption and ossification of cartilage during the development of long bones

During development of the skeleton, osteoclast (OC) recruitment and migration are required for the vascular invasion of the cartilaginous anlage and the ossification of long bones. c-Cbl lies downstream of the vitronectin receptor and forms a complex with c-Src and Pyk2 in a signaling pathway that is required for normal osteoclast motility. To determine whether the decreased motility we observed in vitro in c-Cbl(-/-) OCs translated into decreased cell migration in vivo, we analyzed the long bones of c-Cbl(-/-) mice during development. Initiation of vascularization and replacement of cartilage by bone were delayed in c-Cbl(-/-) mice, due to decreased osteoclast invasion of the hypertrophic cartilage through the bone collar. Furthermore, c-Cbl(-/-) mice show a delay in the formation of secondary centers of ossification, a thicker hypertrophic zone of the growth plate, and a prolonged presence of cartilaginous remnants in the spongiosa, confirming a decrease in resorption of the calcified cartilage. Thus, the decrease in motility of c-Cbl(-/-) osteoclasts observed in vitro results in a decreased ability of osteoclasts to invade and resorb bone and mineralized cartilage in vivo. These results confirm that c-Cbl plays an important role in osteoclast motility and resorbing activity.

Calpain Is Required for Normal Osteoclast Function and Is Down-regulated by Calcitonin *

Osteoclast motility is thought to depend on rapid podosome assembly and disassembly. Both μ-calpain and m-calpain, which promote the formation and disassembly of focal adhesions, were observed in the podosome belt of osteoclasts. Calpain inhibitors disrupted the podosome belt, blocked the constitutive cleavage of the calpain substrates filamin A, talin, and Pyk2, which are enriched in the podosome belt, induced osteoclast retraction, and reduced osteoclast motility and bone resorption. The motility and resorbing activity of μ-calpain-/- osteoclast-like cells were also reduced, indicating that μ-calpain is required for normal osteoclast activity. Histomorphometric analysis of tibias from μ-calpain-/- mice revealed increased osteoclast numbers and decreased trabecular bone volume that was apparent at 10 weeks but not at 5 weeks of age. In vitro studies suggested that the increased osteoclast number in the μ-calpain-/- bones resulted from increased osteoclast survival, not increased osteoclast formation. Calcitonin disrupted the podosome ring, induced osteoclast retraction, and reduced osteoclast motility and bone resorption in a manner similar to the effects of calpain inhibitors and had no further effect on these parameters when added to osteoclasts pretreated with calpain inhibitors. Calcitonin inhibited the constitutive cleavage of a fluorogenic calpain substrate and transiently blocked the constitutive cleavage of filamin A, talin, and Pyk2 by a protein kinase C-dependent mechanism, demonstrating that calcitonin induces the inhibition of calpain in osteoclasts. These results indicate that calpain activity is required for normal osteoclast activity and suggest that calcitonin inhibits osteoclast bone resorbing activity in part by down-regulating calpain activity.

Loss of Cbl-b Increases Osteoclast Bone-Resorbing Activity and Induces Osteopenia

Cbl proteins are multifunctional adaptor molecules that modulate cellular activity by targeting the ubiquitylating system, endocytic complexes, and other effectors to a wide variety of regulatory proteins, especially activated receptor and nonreceptor tyrosine kinases. Cbl and Cbl‐b perform unique functions in various cells, in addition to redundant functions that are required for embryonic development. We previously showed that eliminating Cbl impaired osteoclast motility, which modestly delayed embryonic bone development. We now report that Cbl‐b−/− mice are osteopenic, because of increased bone resorption with little compensating increase in bone formation. In vitro bone‐resorbing activity and differentiation of osteoclast‐like cells (OCLs) were increased, as were some RANKL‐induced signaling events (activation of NF‐κB and the mitogen‐activated protein kinases extracellular signal‐regulated kinase [ERK] and p38), suggesting that specific RANKL‐activated mechanisms contribute to the increased rate of differentiation and bone‐resorbing activity. Re‐expressing Cbl‐b in Cbl‐b−/− OCLs normalized the increased bone‐resorbing activity and overexpressing Cbl‐b in wildtype OCLs inhibited bone resorption. Cbl was without effect in either wildtype or Cbl‐b−/− OCLs. Functional tyrosine kinase binding (TKB) and RING finger domains were required for the rescue by Cbl‐b. Thus, both Cbl and Cbl‐b perform regulatory functions in osteoclasts that are unique to one or the other protein (i.e., functions that cannot be compensated by the other homolog). One of Cbl‐bs unique functions in osteoclasts is to downregulate bone resorption.

Bone protection by estrens occurs through non–tissue-selective activation of the androgen receptor

The use of estrogens and androgens to prevent bone loss is limited by their unwanted side effects, especially in reproductive organs and breast. Selective estrogen receptor modulators (SERMs) partially avoid such unwanted effects, but their efficacy on bone is only moderate compared with that of estradiol or androgens. Estrens have been suggested to not only prevent bone loss but also exert anabolic effects on bone while avoiding unwanted effects on reproductive organs. In this study, we compared the effects of a SERM (PSK3471) and 2 estrens (estren-alpha and estren-beta) on bone and reproductive organs to determine whether estrens are safe and act via the estrogen receptors and/or the androgen receptor (AR). Estrens and PSK3471 prevented gonadectomy-induced bone loss in male and female mice, but none showed true anabolic effects. Unlike SERMs, the estrens induced reproductive organ hypertrophy in both male and female mice and enhanced MCF-7 cell proliferation in vitro. Estrens directly activated transcription in several cell lines, albeit at much higher concentrations than estradiol or the SERM, and acted for the most part through the AR. We conclude that the estrens act mostly through the AR and, in mice, do not fulfill the preclinical efficacy or safety criteria required for the treatment or prevention of osteoporosis.

Doubly Truncated FosB Isoform (Δ2ΔFosB) Induces Osteosclerosis in Transgenic Mice and Modulates Expression and Phosphorylation of Smads in Osteoblasts Independent of Intrinsic AP-1 Activity

Introduction: Activator protein (AP)‐1 family members play important roles in the development and maintenance of the adult skeleton. Transgenic mice that overexpress the naturally occurring ΔFosB splice variant of FosB develop severe osteosclerosis. Translation of Δfosb mRNA produces both ΔFosB and a further truncated isoform (Δ2ΔFosB) that lacks known transactivation domains but, like ΔFosB, induces increased expression of osteoblast marker genes.

Lack of ADAM15 in mice is associated with increased osteoblast function and bone mass.

Abstract The ADAMs (a disintegrin and metalloprotease) contribute to various biological functions including the development of tissues by taking part in cell-cell and cell-matrix interactions. We previously found that ADAM15 is prominently expressed in osteoblasts and to a lesser extent in osteoclasts. The aim of this study was to investigate a possible function of ADAM15 in bone. Adult ADAM15-/- mice displayed an increase in bone volume and thickness with an increase in the number and activity of osteoblasts, whereas osteoclasts were apparently unaffected. We found an increase in proliferation, alkaline phosphatase (ALP) staining and nodule deposition, and mineralization in cultures of ADAM15-/- osteoblasts compared to wild-type osteoblasts. We also observed an increase in β-catenin immunoreactivity in the nucleus of ADAM15-/- osteoblasts compared to wild-type, whereas β-catenin in the membrane/cytoplasm compartment appeared to undergo increased degradation. Furthermore, cyclin D1 and c-Jun, known downstream targets of β-catenin and effectors of cell activation, were found up-regulated in absence of ADAM15. This study indicates that ADAM15 is required for normal skeletal homeostasis and that its absence causes increased nuclear translocation of β-catenin in osteoblasts leading to increased osteoblast proliferation and function, which results in higher trabecular and cortical bone mass.