Silvia Migliaccio

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment

Background: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). Methods: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). Results: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. Conclusions: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

The selective estrogen receptor modulator raloxifene regulates osteoclast and osteoblast activity in vitro

Raloxifene is a selective estrogen receptor modulator (SERM) that prevents bone loss. Although it is largely used for the treatment of osteoporosis, the mechanisms by which this compound modulates the activity of bone cells are still poorly understood. In this study we investigate whether raloxifene affects osteoclast and osteoblast activity in vitro. Bone marrow cultures were established from neonatal mice and treated with 1,25(OH)(2) vitamin D(3) (VitD(3), 10(-8) mol/L) to induce osteoclast generation. Similar to 17beta-estradiol, raloxifene significantly reduced the number of osteoclasts in a concentration-dependent manner, with maximal inhibition at 10(-11) mol/L (-48%). However, as for 17beta-estradiol, at a high concentration (10(-7) mol/L), the inhibitory effect of raloxifene was abolished. In a pit assay, raloxifene inhibited bone resorption. A maximal effect was observed at 10(-9) mol/L, and maintained at a high concentration, indicating that inhibition of osteoclast formation and inhibition of bone resorption may be due to activation of, at least in part, different pathways. Osteoblasts from neonatal mice calvariae were also exposed to raloxifene. In these cells, this compound induced a concentration-dependent increase of proliferation, which was blocked by the estrogen-receptor antagonist ICI 164,384. Raloxifene also increased the osteoblast-specific transcription factor Cbfa1/Runx2 and alpha2 procollagen type I chain mRNAs, with a pattern that only partially coincided with that of 17beta-estradiol. Consistent with decreased osteoclastogenesis, raloxifene inhibited the mRNA expression of interleukin (IL)-1beta and IL-6 at a low concentration, but not at a high concentration, whereas 17beta-estradiol had similar effects on IL-6 and inhibited IL-1beta at both concentrations. Furthermore, both compounds were able to inhibit tumor necrosis factor (TNF)-alpha-induced IL-1beta, but not IL-6, increase. In conclusion, these data show that raloxifene negatively modulates osteoclasts, and positively affects osteoblasts, suggesting not only an antiresorptive role, but also an osteoblast stimulatory role.

Genotype-Phenotype Relationship in Human ATP6i-Dependent Autosomal Recessive Osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.

Decreased Proliferation and Altered Differentiation in Osteoblasts from Genetically and Clinically Distinct Craniosynostotic Disorders

Craniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a number of such conditions. Nevertheless, the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution, Apert syndrome with FGFR2 P253R change, and a nonsyndromic craniosynostosis without FGFR canonic mutations, as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins, associated with high expression and activation of protein kinase Calpha and protein kinase Cepsilon isoenzymes. By contrast, the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control, C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase Czeta levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch, associated with alterations of the FGFR transduction pathways, could be the causative common feature in craniosynostosis and that mutations in distinct FGFR2 domains are associated with an in vitro heterogeneous differentiative phenotype.

is obesity in women protective against osteoporosis

The belief that obesity is protective against osteoporosis has recently come into question. The latest epidemiologic and clinical studies have shown that a high level of fat mass might be a risk factor for osteoporosis and fragility fractures. Further, increasing evidence seems to indicate that different components of the metabolic syndrome, ie, hypertension, increased triglycerides, reduced high-density lipoprotein cholesterol, are also potential risk factors for the development of low bone mineral density and osteoporosis. This review considers both the older and more recent data in the literature in order to evaluate further the relationship between fat tissue and bone tissue.

Effects of long-acting testosterone undecanoate on bone mineral density in middle-aged men with late-onset hypogonadism and metabolic syndrome: results from a 36 months controlled study

We evaluated the effects of long-term testosterone replacement therapy (TRT) on the bone mineral density (BMD) in obese patients with metabolic syndrome (MS) and late-onset hypogonadism (LOH). Sixty men (mean age 57 ± 10) with low serum testosterone (T < 320 ng/dL) and MS regardless the presence of osteoporosis were enrolled. Forty men received intramuscular T-undecanoate (TU) four times/year for 36 months and 20 age-matched hypogonadal men with MS in whom T treatment was contraindicated were used as controls. Hormonal, biochemical markers, vertebral and femoral BMD by dual-energy x-ray absorptiometry were measured. At baseline, overall patients had mild osteopenia (lumbar BMD= 0.891 ± 0.097 g/cm2; femoral BMD= 0.847 ± 0.117 g/cm2). TU induced a significant improvement of bone mass after 36 months (lumbar BMD = 1.053 ± 0.145 g/cm2; p < 0.002; femoral BMD = 0.989 ± 0.109; p < 0.003 g/cm2) with a 5%/year increase and a significant reduction in hs-CRP without changes in body mass index. A direct relationship between serum T and BMD increments at the lumbar (r2 = 0.66, p < 0.0001) and femoral (r2 = 0.52, p < 0.0001) sites was demonstrated. Study adherence was 50% without serious side effects. Long-term TRT in middle-aged men with LOH and MS determines a significant increase in both vertebral and femoral BMD related to increased serum T levels, probably independently from estradiol modifications.

A new heterozygous mutation (R714C) of the osteopetrosis gene, pleckstrin homolog domain containing family M (with run domain) member 1 (PLEKHM1), impairs vesicular acidification and increases TRACP secretion in osteoclasts.

We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast–osteoblast cross‐talk.

The obesity of bone

During the last decades, obesity and osteoporosis have become important global health problems, and the belief that obesity is protective against osteoporosis has recently come into question. In fact, some recent epidemiologic and clinical studies have shown that a high level of fat mass might be a risk factor for osteoporosis and fragility fractures. Several potential mechanisms have been proposed to explain the complex relationship between adipose tissue and bone. Indeed, adipose tissue secretes various molecules, named adipokines, which are thought to have effects on metabolic, skeletal and cardiovascular systems. Moreover, fat tissue is one of the major sources of aromatase, an enzyme that synthesizes estrogens from androgen precursors, hormones that play a pivotal role in the maintenance of skeletal homeostasis, protecting against osteoporosis. Moreover, bone cells express several specific hormone receptors and recent observations have shown that bone-derived factors, such as osteocalcin and osteopontin, affect body weight control and glucose homeostasis. Thus, the skeleton is considered an endocrine target organ and an endocrine organ itself, likely influencing other organs as well. Finally, adipocytes and osteoblasts originate from a common progenitor, a pluripotential mesenchymal stem cell, which has an equal propensity for differentiation into adipocytes or osteoblasts (or other lines) under the influence of several cell-derived transcription factors. This review will highlight recent insights into the relationship between fat and bone, evaluating both potential positive and negative influences between adipose and bone tissue. It will also focus on the hypothesis that osteoporosis might be considered the obesity of bone.

Colony stimulating factor-1-induced osteoclast spreading depends on substrate and requires the vitronectin receptor and the c-src proto-oncogene.

The colony stimulating factor 1 (CSF‐1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF‐1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF‐1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF‐1–induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c‐src proto‐oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF‐1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8‐fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF‐1 induced a 2.5‐fold increase of osteoclasts showing filopodia, and a 9.5‐fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF‐1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF‐1–induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c‐src proto‐oncogene, cells were treated with the αvβ3 integrin neutralizing antibody, LM609, or c‐src antisense oligonucleotides, which reduced CSF‐1–induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF‐1–induced osteoclast spreading requires both the vitronectin receptor and the c‐src proto‐oncogene and that this action is modulated by the adhesion substrata.

Mechanisms of Osteoclast Dysfunction in Human Osteopetrosis: Abnormal Osteoclastogenesis and Lack of Osteoclast-Specific Adhesion Structures

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate‐resistant acid phosphatase (TRAP)‐positive, multinucleated and mononuclear cells, and alkaline phosphatase‐positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP‐positive precursors spontaneously fused in culture to form giant osteoclast‐like cells. These cells expressed the osteoclast marker MMP‐9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c‐src, c‐fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP‐positive multinucleated cells did not form osteoclast‐specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast‐like cells.