Addolorata Corrado

Osteoblast physiology in normal and pathological conditions

Osteoblasts are mononucleated cells that are derived from mesenchymal stem cells and that are responsible for the synthesis and mineralization of bone during initial bone formation and later bone remodelling. Osteoblasts also have a role in the regulation of osteoclast activity through the receptor activator of nuclear factor κ-B ligand and osteoprotegerin. Abnormalities in osteoblast differentiation and activity occur in some common human diseases such as osteoporosis and osteoarthritis. Recent studies also suggest that osteoblast functions are compromised at sites of focal bone erosion in rheumatoid arthritis.

Osteocalcin: Skeletal and extra‐skeletal effects

Osteocalcin (OC) is a non‐collagenous, vitamin K‐dependent protein secreted in the late stage of osteoblasts differentiation. The presence of the three residues of γ‐carbossiglutamatic acid, specific of the active form of OC protein, allows the protein to bind calcium and consequently hydroxyapatite. The osteoblastic OC protein is encoded by the bone γ‐carbossiglutamate gene whose transcription is principally regulated by the Runx2/Cbfa1 regulatory element and stimulated by vitamin D3 through a steroid‐responsive enhancer sequence. Even if data obtained in literature are controversial, the dual role of OC in bone can be presumed as follows: firstly, OC acts as a regulator of bone mineralization; secondly, OC regulates osteoblast and osteoclast activity. Recently the metabolic activity of OC, restricted to the un‐carboxylated form has been demonstrated in osteoblast‐specific knockout mice. This effect is mediated by the regulation of pancreatic β‐cell proliferation and insulin secretion and adiponectin production by adipose tissue and leads to the regulation of glucose metabolism and fat mass. Nevertheless, clinical human studies only demonstrated the correlation between OC levels and factors related to energy metabolism. Thus further investigations in humans are required to demonstrate the role of OC in the regulation of human energy metabolism. Moreover, it is presumable that OC also acts on blood vessels by inducing angiogenesis and pathological mineralization. This review highlights the recent studies concerning skeletal and extra‐skeletal effects of OC. J. Cell. Physiol. 228: 1149–1153, 2013.

Lymphocytes and synovial fluid fibroblasts support osteoclastogenesis through RANKL, TNFα, and IL‐7 in an in vitro model derived from human psoriatic arthritis

Psoriatic arthritis (PsA) is an inflammatory joint disease, characterized by extensive bone resorption, whose mechanisms have not been fully elucidated. Thus, in the present study we investigated the involvement of RANKL, TNFα, and IL‐7 in the osteoclastogenesis of PsA patients. In vitro osteoclastogenesis models, consisting of unfractionated and T‐cell‐depleted mononuclear cells from peripheral blood (PBMCs) and synovial fluid (SFMCs) of 20 PsA patients as well as from healthy donors were studied. Freshly isolated T and B cells from PBMCs and T cells and fibroblasts from SFMCs of PsA patients were subjected to RT‐PCR to detect the levels of RANKL, TNFα, and IL‐7. Osteoclastogenesis was studied in the presence of RANK‐Fc, anti‐TNFα, and anti IL‐7 functional antibodies. We demonstrate that lymphocytes and fibroblasts support osteoclast (OC) formation in PsA patients through the production of osteoclastogenic cytokines. In particular, OC formation was completely abolished in unstimulated T cell‐depleted PBMC cultures, and reduced by approximately 70% in unstimulated T cell‐depleted SFMC cultures. Freshly isolated T cells from PBMCs and SFMCs of PsA patients overexpressed RANKL and TNFα, while fibroblasts from synovial fluid produced only RANKL. We show that the presence of RANK‐Fc and/or anti‐TNFα functional antibodies reduced OC formation. Moreover, T and B cells from PBMCs as well as T cells and fibroblasts from SFMCs expressed IL‐7 mRNA. Finally, the anti‐IL‐7 functional antibody significantly reduced osteoclastogenesis. Our results suggest that fibroblasts, B and T lymphocytes support OC formation by producing RANKL, TNFα, and IL‐7, contributing to the aggressive bone resorption in PsA patients. Copyright

Bisphosphonates: effects on osteoblast

PurposeBisphosphonates are synthetic analogues of pyrophosphate usually used in treating bone disorders such as osteoporosis, Paget’s disease, fibrous dysplasia, hypercalcemia of malignancy, and inflammation-related bone loss. Though therapeutic effects of bisphosphonates depend primarily on their inhibitory effect on osteoclasts, increasing attention is being given to other effector cells, such as osteoblasts. This review focuses on the presumed effect of bisphosphonates on osteoblasts.MethodsA review of the literature was conducted to evaluate the pharmacodynamic effects of bisphosphonates including inhibition of osteoclasts and apoptosis of osteocytes and osteoblasts as well as their potential stimulatory effects on the proliferation of osteoblasts.ResultsStudies have demonstrated that bisphosphonates may stimulate proliferation of osteoblasts and inhibit apoptosis of osteocytes and osteoblasts.ConclusionConsidering that osteoblasts may be involved in bone disorders, such as osteoporosis, osteopetrosis, osteogenesis imperfecta, and Paget’s disease, and that bisphosphonates may stimulate proliferation of osteoblasts and inhibit apoptosis of osteocytes and osteoblasts, it is conceivable that a role for bisphosphonates exists in these diseases beyond merely the osteoclast influence.

Osteocytes: central conductors of bone biology in normal and pathological conditions.

Osteocytes are the most abundant and longest‐living cells in the adult skeleton. For a long time, osteocytes were considered static and inactive cells, but in recent years, it has been suggested that they represent the key responder to various stimuli that regulate bone formation and remodelling as well as one of the key endocrine regulators of bone metabolism. Osteocytes respond to mechanical stimuli by producing and secreting several signalling molecules, such as nitric oxide and prostaglandin E2, that initiate local bone remodelling. Moreover, they can control bone formation by modulating the WNT signalling pathway, an essential regulator of cell fate and commitment, as they represent the main source of sclerostin, a negative regulator of bone formation. Osteocytes can also act as an endocrine organ by releasing fibroblast growth factor 23 and several other proteins (DMP‐1, MEPE, PHEX) that regulate phosphate metabolism. It has been demonstrated that various bone diseases are associated with osteocyte abnormalities, although it is not clear if these changes are the direct cause of the pathology or if they are secondary to the pathological changes in the bone microenvironment. Thus, a better understanding of these cells could offer exciting opportunities for new advances in the prevention and management of different bone diseases.

Systemic effects of Wnt signaling

Wnt signaling plays a key role in several physiological and pathological aspects. Even if Wnt signal was first described more than 20 years ago, its role in systemic effects, such as angiogenesis and vascular disorders, bone biology, autoimmune diseases, neurological diseases, and neoplastic disorders, was only recently emerged through the use of animal and in vitro models. Moreover, Wnt signaling inhibitors, such as DKK‐1, may be advantageously considered targets for the treatment of several diseases, including osteoporosis, vascular diseases, inflammatory diseases, neurological diseases, and cancer. Nevertheless, further studies are required to provide a complete understanding of this complex signaling pathway, and especially of its role in human diseases, considering the possible advantageous effects of Wnt signaling inhibitors on the progression of disease conditions. J. Cell. Physiol. 228: 1428–1432, 2013.

Extracellular matrix modulates angiogenesis in physiological and pathological conditions.

Angiogenesis is a multistep process driven by a wide range of positive and negative regulatory factors. Extracellular matrix (ECM) plays a crucial role in the regulation of this process. The degradation of ECM, occurring in response to an angiogenic stimulus, leads to degradation or partial modification of matrix molecules, release of soluble factors, and exposure of cryptic sites with pro- and/or antiangiogenic activity. ECM molecules and fragments, resulting from proteolysis, can also act directly as inflammatory stimuli, and this can explain the exacerbated angiogenesis that drives and maintains several inflammatory diseases. In this review we have summarized some of the more recent literature data concerning the molecular control of ECM in angiogenesis in both physiological and pathological conditions.

Osteocalcin synthesis by human osteoblasts from normal and osteoarthritic bone after vitamin D3 stimulation

Alterations in osteoblast metabolism are involved in the pathogenesis of typical subchondral bone changes in osteoarthritis (OA). Osteocalcin is a specific bone protein, synthesised by the osteoblasts, which can be considered a marker of metabolic activity of these cells. In this study we correlated osteocalcin production from human osteoblasts isolated from healthy and osteoarthritic subjects to the degree of cartilage damage, before and after stimulation with 1,25(OH)2-vitamin D3, the active metabolite of vitamin D3. We isolated human osteoblasts from cancellous bone of healthy subjects and from subchondral bone of osteoarthritic subjects and considered the osteoblasts corresponding to different degrees of cartilage damage as different cell populations. We determined the osteocalcin production in normal and osteoarthritic osteoblasts from maximal and minimal cartilage damage areas both under basal conditions and after vitamin D3 stimulation. Compared to normal osteoblasts, under basal conditions osteocalcin production is significantly greater in osteoarthritic osteoblasts, corresponding both to maximal and minimal damage joint areas. No differences were observed between osteoblasts from maximal and minimal damage areas. The response of osteoblasts to vitamin D3 stimulation appeared to be proportional to the degree of joint damage, as the vitamin D3-induced increase in osteocalcin is proportionally greater in maximally damaged osteoblasts compared to minimally damaged ones. Thus, after vitamin D3 stimulation, a significant increase in osteocalcin production by maximally damaged osteoblasts compared to the minimally damaged ones was observed. This study confirms abnormal osteoarthritic osteoblast behaviour and indicates that osteoblasts from different areas of the same affected joint may be metabolically different, supporting the hypothesis that subchondral osteoblasts may play an essential role in the pathogenesis of OA.

Osteoporosis and rheumatic diseases.

Numerous rheumatic diseases, including rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, systemic sclerosis, dermatomyositis/polymyositis and vasculitis are characterized by osteoporosis and fragility fractures. Inflammatory cytokines, glucocorticoid treatment, immobilization and reduced physical activity due to painful joints and muscle weakness are considered the main risk factors that cause low body mass density values in these diseases. Emerging evidence highlights the role of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-7 and IL-17, in the regulation of the bone homeostasis. In fact, chronic inflammation is often characterized by an imbalance between bone formation and bone resorption with a net prevalence of osteoclastogenesis, which is an important determinant of bone loss in rheumatic diseases.

RANKL/OPG Ratio and DKK-1 Expression in Primary Osteoblastic Cultures from Osteoarthritic and Osteoporotic Subjects

Objective. To evaluate the expression of Dickkopf-1 protein factor (DKK-1), DKK-2, and β-catenin, components of the Wnt pathway, in human osteoarthritic (OA) and osteoporotic (OP) osteoblasts and to correlate it to cell metabolic activity, proliferation, and receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) expression. Methods. Primary human osteoblast cultures were obtained from healthy, OA, and OP donors. In each cell population we evaluated DKK-1, DKK-2, nonphosphorylated β-catenin and RANKL/OPG expression, osteocalcin and alkaline phosphatase (ALP) synthesis, and cell proliferation, both in basal condition and after vitamin D3 stimulation. Results. DKK-1 and DKK-2 showed opposite patterns of expression in OA and OP osteoblasts. The RANKL/OPG ratio was significantly higher in the OP group because of a greater expression of RANKL, whereas it was significantly lower in the OA group because of a higher expression of OPG. Treatment with vitamin D3 increased the RANKL/OPG ratio and DKK-2 expression and reduced DKK-1 expression in each cell population, but did not affect β-catenin levels. Both osteocalcin and ALP production and cell proliferation were enhanced in OA cells and reduced in the OP ones. Conclusion. These data confirm that OA and OP are characterized by opposite bone changes, consisting of reduced bone remodeling processes with increased osteoblast activity in OA, and enhanced bone resorptive activity with reduction of osteoblast metabolism in OP, and suggest that the Wnt pathway is involved in the pathogenesis of both diseases.