Bone Cell Physiology Research Articles

Bone-Targeted Agents and Metastasis Prevention

Simple SummaryBone metastases are common in cancer patients and the role of bone-targeted agents in prevention of metastasis have been evaluated in multiple clinical trials over the past 20 years. Results show that in breast cancer the use of adjuvant bisphosphonates (and possibly denosumab) reduce bone metastases and breast cancer deaths in postmenopausal women. These effects are in addition to the benefits associated with the use of standard adjuvant endocrine, cytotoxic and targeted treatments with prevention of one in six breast cancer deaths at 10 years. Similar benefits have not been observed in other cancers that typically spread to bone, such as prostate or lung cancers. Biomarkers that can predict patient benefit from the use of bone targeted treatments in the adjuvant setting are being evaluated. Currently, tumour expression of the transcription factor, MAF, seems to be the most promising biomarker; benefits from adjuvant bisphosphonates are seen in the 80% of patients with normal levels of expression irrespective of menopausal status, while over-expression is associated with a poor prognosis and a higher rate of visceral metastases.The use of bone-targeted treatments has transformed the clinical care of many patients with metastatic breast cancer. In addition, due to the profound effects of bisphosphonates and denosumab on bone physiology and the bone microenvironment, the potential of bone-targeted agents to modify the process of metastasis has been studied extensively. Many adjuvant trials with bisphosphonates in early breast cancer have been performed. Variable outcomes in terms of disease recurrence have been reported, with any treatment benefits apparently influenced by the age and menopausal status of the patients. The Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) conducted a meta-analysis of individual patient data from all available randomised trials to investigate this observation further. This meta-analysis failed to show any benefits of adjuvant bisphosphonates in premenopausal women, but highly significant improvements in bone recurrence (RR = 0.72; 95% CI 0.60–0.86, 2p = 0.0002) and breast cancer mortality (RR = 0.82; 95% CI 0.73–0.93, 2p = 0.002) were seen in the 11,767 postmenopausal women included in the meta-analysis. As a result, clinical guidelines recommend the incorporation of adjuvant bisphosphonates that inhibit osteoclast activity into routine clinical care. Denosumab, which has similar effects on bone cell physiology, appears not to consistently influence disease outcomes, perhaps suggesting that it is the “off target” effects of bisphosphonates on immune function and the biological processes involved in metastasis that are important. Predictive biomarkers beyond menopause are being sought and assessment of the transcription factor MAF (mesenchymal aponeurotic fibrosarcoma gene) appears to identify patients able to benefit from the addition of a bisphosphonate to standard adjuvant anticancer therapies.

Tetracyclines and bone: Unclear actions with potentially lasting effects.

Tetracyclines are a broad-spectrum class of antibiotics that have unclear actions with potentially lasting effects on bone metabolism. Initially isolated from Streptomyces, tetracycline proved to be an effective treatment for Gram +/- infections. The emergence of resistant bacterial strains commanded the development of later generation agents, including minocycline, doxycycline, tigecycline, sarecycline, omadacycline, and eravacycline. In 1957, it was realized that tetracyclines act as bone fluorochrome labels due to their high affinity for the bone mineral matrix. Over the course of the next decade, researchers discerned that these compounds are retained in the bone matrix at high levels after the termination of antibiotic therapy. Studies during this period provided evidence that tetracyclines could disrupt prenatal and early postnatal skeletal development. Currently, tetracyclines are most commonly prescribed as a long-term systemic therapy for the treatment of acne in healthy adolescents and young adults. Surprisingly, the impact of tetracyclines on physiologic bone modeling/remodeling is largely unknown. This article provides an overview of the pharmacology of tetracycline drugs, summarizes current knowledge about the impact of these agents on skeletal development and homeostasis, and reviews prior work targeting tetracyclines' effects on bone cell physiology. The need for future research to elucidate unclear effects of tetracyclines on the skeleton is addressed, including drug retention/release mechanisms from the bone matrix, signaling mechanisms at bone cells, the impact of newer third generation tetracycline antibiotics, and the role of the gut-bone axis.

Extracellular Vesicles From Osteotropic Breast Cancer Cells Affect Bone Resident Cells.

Extracellular vesicles (EVs) are emerging as mediators of a range of pathological processes, including cancer. However, their role in bone metastases has been poorly explored. We investigated EV-mediated effects of osteotropic breast cancer cells (MDA-MB-231) on bone resident cells and endothelial cells. Pretreatment of osteoblasts with conditioned medium (CM) of MDA-MB-231 (MDA) cells promoted pro-osteoclastogenic and pro-angiogenic effects by osteoblast EVs (OB-EVs), as well as an increase of RANKL-positive OB-EVs. Moreover, when treating osteoblasts with MDA-EVs, we observed a reduction of their number, metabolic activity, and alkaline phosphatase (Alp) activity. MDA-EVs also reduced transcription of Cyclin D1 and of the osteoblast-differentiating genes, while enhancing the expression of the pro-osteoclastogenic factors Rankl, Lcn2, Il1b, and Il6. Interestingly, a cytokine array on CM from osteoblasts treated with MDA-EVs showed an increase of the cytokines CCL3, CXCL2, Reg3G, and VEGF, while OPG and WISP1 were downregulated. MDA-EVs contained mRNAs of genes involved in bone metabolism, as well as cytokines, including PDGF-BB, CCL3, CCL27, VEGF, and Angiopoietin 2. In line with this profile, MDA-EVs increased osteoclastogenesis and in vivo angiogenesis. Finally, intraperitoneal injection of MDA-EVs in mice revealed their ability to reach the bone microenvironment and be integrated by osteoblasts and osteoclasts. In conclusion, we showed a role for osteoblast-derived EVs and tumor cell-derived EVs in the deregulation of bone and endothelial cell physiology, thus fueling the vicious cycle induced by bone tumors. © 2019 American Society for Bone and Mineral Research.

Metabolic Bone Diseases and Total Hip Arthroplasty: Preventing Complications.

Metabolic bone diseases are a diverse group of conditions characterized by abnormalities in calcium metabolism and/or bone cell physiology. These unbalanced processes can eventually lead to bony deformities and altered joint biomechanics, resulting in degenerative joint disease. Not infrequently, patients with metabolic bone diseases have restricting hip joint pain that ultimately necessitates hip arthroplasty. To minimize complications, the surgeon must consider the particular characteristics of these patients. The surgical and medical management of patients with metabolic bone diseases undergoing hip arthroplasty requires appropriate preoperative diagnosis, careful attention to the technical challenges of surgery, and strategies to maximize the long-term results of the surgical intervention, such as the use of bone anabolic and anticatabolic agents.

Bone demineralization is improved by ivacaftor in patients with cystic fibrosis carrying the p.Gly551Asp mutation

Low bone mineral density (BMD) is a common problem in adults with cystic fibrosis (CF), the etiology of which is multifactorial. In this study, we provide the first evidence that ivacaftor improves BMD in CF patients carrying the p.Gly551Asp mutation. Consistently, in vitro experiments with TNF-α-stimulated primary F508del-CFTR osteoblasts demonstrated that correction of p.Phe508del-CFTR markedly decreased RANKL protein production, a major factor of bone resorption. These clinical and fundamental observations suggest that rescue of mutated CFTR protein improves bone remodeling and support the link between CFTR and bone cell physiology. These findings represent a step forward in the development of potential new therapies for CF-related bone disease.

In vitro three-dimensional bone tissue models: from cells to controlled and dynamic environment.

Most of our knowledge of bone cell physiology is derived from experiments carried out in vitro on polystyrene substrates. However, these traditional monolayer cell cultures do not reproduce the complex and dynamic three-dimensional (3D) environment experienced by cells in vivo. Thus, there is a growing interest in the use of 3D culture systems as tools for understanding bone biology. These in-vitro-engineered systems, less complex than in vivo models, should ultimately recapitulate and control the main biophysical, biochemical, and biomechanical cues that define the in vivo bone environment, while allowing their monitoring. This review focuses on state-of-the-art and the current advances in the development of 3D culture systems for bone biology research. It describes more specifically advantages related to the use of such systems, and details main characteristics and challenges associated with its three main components, that is, scaffold, cells, and perfusion bioreactor systems. Finally, future challenges for noninvasive imaging technologies are addressed.

FRI0048 Hypoxia in human osteoarthritic subchondral bone: a risk factor for leptin production by osteoblasts

BackgroundClinical and in vitro studies suggest that subchondral bone is involved in the progression and/or onset of osteoarthritis (OA). Human osteoblasts (Obs) isolated from subchondral OA bone tissue show an...

Collagen osteoid-like model allows kinetic gene expression studies of non-collagenous proteins in relation with mineral development to understand bone biomineralization.

Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT- and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.

Expression of β-Catenin and γ-Catenin in Maxillary Bone Regeneration

β- and γ-catenin are components of catenin family involved in cadherin adhesion function. Recently it has been shown that this family is involved in other functions such as signaling and activation of transcription factors. The final goal of this study was to evaluate the role of β- and γ-catenin in bone cell physiology and bone regeneration. Formalin-fixed-paraffin embedded specimens of 15 human bone specimens after sinus lift were collected and examined by immunohistochemstry using primary antibodies against β- and γ-catenin. Staining intensity and cellular localization were evaluated. β and γ-catenin showed a very high level of expression in human bone tissues. In particular catenins were expressed in cells with morphological findings of osteoblasts in the areas of new bone formation at the junction between mineralized and unmineralized tissue, between osteoid matrix and bone. Osteoclasts showed also positivity for catenins. Osteocytes, cells located in lacunae of mature mineralized bone with function of bone vitality maintenance, showed no expression for catenins. Specimens characterized by high amount of catenins in osteoblasts at 1° month showed high grade of bone maturation at 3° month. Data demonstrated an overall involvement of catenins in human bone tissues and in particular during bone regeneration process. The presence of staining for β- and γ-catenin particularly in osteoblasts demonstrates a significant role of catenins in functions, other than in cadherin interaction, such as signaling and activation of transcription factors during differentiation of bone tissues.

A remarkable role of growth factors in resolving oral and specific periodontal pathologies: A strategic review

The knowledge and the understanding of the role of growth factors, their mechanisms of action, and molecular signaling pathways, which have been reviewed in this article, suggest the potential for many novel therapeutic targets, not only for applying growth factors but also for the potential use of growth factor inhibitors or agents that target specific parts of the intracellular signaling pathways in controlling oral pathologies. There remains an enormous challenge to convert some of the knowledge from basic studies of bone cell physiology and inflammatory cells to therapeutically useful techniques for the future.

Prostaglandin expression profile in hypoxic osteoblastic cells

Conditions such as fracture and unloading have been shown to be associated with tissue and cellular hypoxia in bone. The effects of hypoxia on bone cell physiology and ultimately its impact on bone tissue repair and remodeling are not well understood. In this study, we investigated the role of hypoxia on prostaglandin release from osteoblastic cells cultured in 2% (hypoxia), 5% (potentially cellular normoxia), and 21% (normoxia for standard cell culture conditions) oxygen for up to 24 h. We quantified the effects of reduced oxygen tension on the release of prostaglandin (PG)E(2), PGF(2alpha), PGD(2), and PGI(2). The mechanism by which hypoxia increases PG production was investigated by examining the various regulatory components of the PG biosynthetic pathway. Our data show that PGE(2) levels alone are significantly elevated under hypoxic conditions. Also, we show that cyclooxygenase (COX)-1 and COX-2 play an important role in hypoxia-induced PGE(2) production, possibly via a mechanism involving changes in their respective activity levels under low oxygen conditions. The effect of hypoxia on PGE(2) levels was mimicked by dimethyloxaloglycine, a known activator of the HIF pathway. In addition, we confirmed that HIF-1alpha was stabilized in osteoblastic cells under hypoxia. Taken together these data suggest a role for the HIF pathway in regulation of PGE(2) levels under hypoxic conditions. Previous studies have detected release of prostaglandins from areas of damaged bone, such as a fracture site, and our data may contribute to an understanding of how this release is regulated.

1,25(OH)2D3 blunts hormone-elevated cytosolic Ca2+ in osteoblast-like cells.

Cytosolic free calcium ([Ca2+]i) is an important regulator of bone cell physiology. We studied the interaction of vitamin D metabolites on the hormonal-activated Ca message system in the osteoblastic cell line UMR-106. The acute rise in [Ca2+]i induced by different calciotropic hormones [parathyroid hormone, prostaglandin E2 (PGE2)] was dose dependently blunted by 1,25-dihydroxyvitamin D [1,25(OH)2D3; half-maximal inhibitory concn approximately 5 x 10(-11) M] and was initially observed after 8 h of preincubation. The 1,25(OH)2D3 metabolite of vitamin D was two orders of magnitude more potent than 24,25(OH)2D3 and 25(OH)D3. To discern between an effect of 1,25(OH)2D3 on hormonal-induced Ca2+ entry through the plasma membrane channel vs. release of Ca2+ from internal stores, we suspended fura-2-loaded cells in Mn2+ rather than Ca2+ buffers. In cells preincubated with 1,25(OH)2D3, [Ca2+]i release (indicated by [Ca2+]i transient) was significantly blunted, whereas Mn2+ influx (indicating Ca2+ flux across the plasma membrane) was unaltered, suggesting a selective effect of 1,25(OH)2D3 on hormonal-activated release of Ca2+ from intracellular stores. 1,25(OH)2D3 also inhibited the PGE2-induced production of inositol 1,4,5-trisphosphate. We conclude that, in osteoblasts, chronic (hours) incubation with 1,25(OH)2D3 leads to attenuated stimulation of the [Ca2+]i transduction pathway by calciotropic hormones. This effect of 1,25(OH)2D3 may provide a cellular basis for the synergism between the effects of vitamin D and calciotropic hormones at the bone level.

Regulation of pregnancy-associated plasma protein-A expression in cultured human osteoblasts

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase secreted by cultured human osteoblasts that has been implicated in the regulation of local insulin-like growth factor (IGF) bioavailability during bone growth and remodeling. However, very little is known about the regulation of PAPP-A expression in bone. In this study, we determined the effect of systemic and local osteoregulatory factors on PAPP-A mRNA and protein expression in normal human osteoblasts (hOB cells). Treatment of hOB cells with particular peptide growth factors (basic fibroblast growth factor, epidermal growth factor), steroid hormones (dexamethasone, 1,25-dihydroxyvitamin D 3), and cytokines [interleukin-6 (IL-6), IL-13, oncostatin M] with known involvement in bone cell physiology had no significant effect on PAPP-A expression. Agents that increase intracellular cyclic AMP (forskolin, prostaglandin E 2) increased PAPP-A mRNA and protein expression ∼3-fold. Tumor necrosis factor α (TNFα), IL-1β, and IL-4 also increased PAPP-A expression 3- to 4-fold. Transforming growth factor β (TGFβ) was previously shown to stimulate PAPP-A expression in hOB cells. The effects of TGFβ, TNFα, and IL-1β were additive, whereas the effects of TGFβ and IL-4 were synergistic. In summary, TNFα, IL-1β, and IL-4 were identified as potent stimulators of PAPP-A expression in primary cultures of human osteoblasts. These findings suggest a mechanism whereby cytokines present in bone and bone marrow could augment IGF bioavailability during skeletal growth and remodeling.

Biology of bone and how it orchestrates the form and function of the skeleton.

The principal role of the skeleton is to provide structural support for the body. While the skeleton also serves as the body's mineral reservoir, the mineralized structure is the very basis of posture, opposes muscular contraction resulting in motion, withstands functional load bearing, and protects internal organs. Although the mass and morphology of the skeleton is defined, to some extent, by genetic determinants, it is the tissue's ability to remodel--the local resorption and formation of bone--which is responsible for achieving this intricate balance between competing responsibilities. The aim of this review is to address bone's form-function relationship, beginning with extensive research in the musculoskeletal disciplines, and focusing on several recent cellular and molecular discoveries which help understand the complex interdependence of bone cells, growth factors, physical stimuli, metabolic demands, and structural responsibilities. With a clinical and spine-oriented audience in mind, the principles of bone cell and molecular biology and physiology are presented, and an attempt has been made to incorporate epidemiologic data and therapeutic implications. Bone research remains interdisciplinary by nature, and a deeper understanding of bone biology will ultimately lead to advances in the treatment of diseases and injuries to bone itself.

Alterations in insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis in transformed osteoblastic cells.

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is secreted by a variety of osteoblastic cells and appears to be an integral component of bone cell physiology. We have previously reported that normal human osteoblast-like (hOB) cells secrete IGFBP-4 as well as a novel IGFBP-4 protease, which requires IGF for functional activity. In this study we assessed the IGFBP-4/IGFBP-4 protease system in transformed osteoblastic cells by Western ligand blotting and cell-free IGFBP-4 protease assays. Simian virus-40-immortalized hOB cells (HOBIT), human osteosarcoma cells (TE-85), and rat osteosarcoma cells (UMR 106-01, ROS 17/2.8) secrete IGFBP-4. In contrast to the rapid and dramatic proteolysis in hOB medium, medium conditioned by these cells had no apparent IGFBP-4 protease activity when assayed with exogenous IGF-II in culture or under cell-free conditions. Assayed in the presence of exogenous protease. HOBIT cells, but not the osteosarcoma cell lines, appeared to produce a cycloheximide-sensitive inhibitor of the IGFBP-4 proteolytic reaction. Transient cell transformation induced by incubating human osteoblasts transfected with a temperature-sensitive mutant of simian virus-40 T-antigen at the permissive temperature or by treating hOB cells with phorbol ester tumor promoters also resulted in inhibition of IGF-dependent IGFBP-4 proteolysis. Inhibition was observed if phorbol ester was added to the cultures at the time of medium change or after the protease had been expressed and secreted. Differences in IGFBP-4 proteolysis could not be accounted for by changes in IGFBP-4 messenger RNA expression or substrate levels. These data suggest that transformation is associated with alterations in the IGFBP-4/IGFBP-4 protease system in osteoblastic cells. Normal human osteoblasts secrete an IGF-dependent IGFBP-4 protease. The induction of an inhibitor of the IGF-dependent IGFBP-4 proteolytic reaction may be associated with early transformation processes. Fully tumorigenic bone cells expressed neither IGFBP-4 protease nor protease inhibitor activity.

Bone cell function, regulation, and communication: A role for nitric oxide

A large array of factors serve as vital communication links between cells and the characterization, regulation, and mechanisms of action of such factors are topics of intense research efforts. Most intercellular messenger molecules which have been described over the years are represented by proteins, small peptides, amino acids or their derivatives, ions, lipid metabolites, or steroids. However, a small uncharged free radical, nitric oxide, has recently garnered much attention as a potent multifunctional signal molecule with widespread actions within and between diverse tissues. Biochemical, molecular, and regulatory studies of the family of enzymes responsible for nitric oxide synthesis, nitric oxide synthases, have established that there are at least three distinct isoforms of this enzyme which are differentially expressed and regulated in various cells or tissues. Modulation of these isoenzyme levels or activities by diverse signals is mediated via transcriptional, translational, and/or post-translational mechanisms, and consequently, alterations in such control may influence normal or pathological processes. Nitric oxide appears to exert pronounced effects on skeletal physiology and its production by various bone cells, elicited target cell responses, modulation by other signalling molecules (e.g., cytokines, hormones, fatty acid derivatives), and chemical interactions with other free radicals (e.g., superoxide anions, hydroxyl radicals) may form one important facet of the many complicated communication pathways controlling bone cell physiology and remodeling. Further cell and molecular studies are needed to address the precise roles that nitric oxide plays in bone development and in the formation and degradation of bone during ordinary bone metabolism. In addition, alterations in the regulation and action of the bone nitric oxide system as a function of certain bone disorders may be manifested by perturbations in bone integrity or mineral homeostasis. In this article, we review the current evidence implicating nitric oxide as an important messenger molecule in bone intercellular communication, speculate on potential roles for this radical in bone biology, and discuss possible future directions for advanced research into the function of nitric oxide in skeletal physiology.

Second messenger signaling of c‐fos gene induction by parathyroid hormone (PTH) and PTH‐related peptide in osteoblastic osteosarcoma cells: Its role in osteoblast proliferation and osteoclast‐like cell formation

The present study was performed to clarify second messenger signaling in parathyroid hormone (PTH)-induced c-fos gene expression, to characterize the participation of the c-fos gene in the regulation of osteoblast proliferation and function as well as osteoclast-like cell formation by PTH and to compare these effects of PTH with those of PTH-related peptide (PTHrP). Both human (h) PTH-(1-34) and hPTHrP-(1-34) at 10(-8) M induced a transient c-fos gene expression to a similar degree in osteoblastic osteosarcoma cells, UMR-106. N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) as well as Sp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Sp-cAMPS), an activator of cAMP-dependent protein kinase (PKA), induced a weak c-fos gene expression. Although Rp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), an inhibitor of PKA, almost completely antagonized dbCAMP- and Sp-cAMPS-induced expression of c-fos gene, it did not cause an obvious inhibition of PTH- or PTHrP-induced expression. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induced an intense expression of the c-fos gene, while 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), incapable of activating PKC, and calcium ionophores (A23187 and ionomycin) did not. Protein kinase C inhibitor (H-7, 50 microM) completely blocked the expression of the c-fos gene by PTH as well as by PTHrP). Antisense oligodeoxynucleotides (as-ODN) complementary to c-fos mRNA, which have been shown to inhibit its mRNA translation, at 1 microM significantly antagonized PTH- and PTHrP-induced inhibition of [3H] thymidine incorporation and stimulation of osteoclast-like cell formation in the presence of osteoblasts, but not an increase in alkaline phosphatase activity, compared to control oligodeoxynucleotides with same nucleotides as as-ODN but with a random sequence. The present study indicates the involvement of PKC system in c-fos gene expression by PTH as well as PTHrP and also indicates the involvement of the c-fos gene in the regulation of bone cell physiology by PTH and PTHrP.

Role of vascular endothelial cells in bone biology.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts, bone-degrading osteoclasts, and other cells present within the bone microenvironment. Balanced control of bone formative and degradative processes is normally carefully maintained in the adult skeleton but becomes uncoupled in the course of aging or in various pathological disease states. Systemic regulators of bone metabolism and local mediators, including matrix molecules, cytokines, prostaglandins, leukotrienes, and other autocrine or paracrine factors, regulate the recruitment, differentiation, and function of cells participating in bone formation and turnover. Although some of these interactions are now understood, many yet remain to be elucidated. Recent studies have begun exploring in detail how vascular endothelial cells and their products function in bone physiology. The findings are revealing that bone vascular endothelial cells may be members of a complex communication network in bone which operates between endothelial cells, osteoblasts, osteoclasts, macrophages, stromal cells, and perhaps other cell types found in bone as well. Therefore, multiple systemic and locally produced signals may be received, transduced, and integrated by individual cells and then propagated by the release from these cells of further signals targeted to other members of the bone cell network. In this manner, bone cell activities may be continuously coordinated to afford concerted actions and rapid responses to physiological changes. The bone microvasculature may play a pivotal role in these processes, both in linking circulatory and local signals with cells of the bone microenvironment and in actively contributing itself to the regulation of bone cell physiology.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of insulin-like growth factor binding protein 4 by a specific insulin-like growth factor binding protein 4 proteinase in normal human osteoblast-like cells: implications in bone cell physiology.

Insulin-like growth factor binding protein 4 (IGFBP-4) is secreted by normal human osteoblast-like cells (hOB) and is a potent inhibitor of insulin-like growth factor (IGF) action in vitro. In previous studies, IGF treatment of hOB in culture led to markedly reduced medium levels of IGFBP-4 as detected by western ligand blotting. In the present study, incubation of hOB-conditioned medium (hOB-CM) with IGF under cell-free conditions resulted in a similar loss of IGFBP-4. Both IGF-I and IGF-II were capable of inducing a decrease in IGFBP-4; however, IGF-II was more effective. When the six characterized IGFBP were added to hOB-CM, only IGFBP-4 disappeared in response to IGF-II addition. This IGF-regulated loss of IGFBP-4 was inhibited by metalloproteinase inhibitors and appeared to be due to a proteinase that cleaved IGFBP-4 in 18 and 14 kD fragments identified by western immunoblotting. Conditioned media from eight of eight different donor hOB lines tested exhibited IGFBP-4 proteinase activity. To assess the biologic consequences of IGF-II-induced IGFBP-4 proteolysis, we treated hOB with IGF-II for 5 h, which decreased medium IGF-BP-4 by 70%, and then measured IGF-I and insulin stimulation of [3H]thymidine incorporation. IGF-II itself was not mitogenic and had no effect on insulin-stimulated [3H]thymidine incorporation. However, pretreatment of cultured hOB with IGF-II enhanced IGF-I-stimulated [3H]thymidine incorporation threefold.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25(OH)2D3 blunts hormone-elevated cytosolic Ca2+ in osteoblast-like cells

Cytosolic free calcium ([Ca2+]i) is an important regulator of bone cell physiology. We studied the interaction of vitamin D metabolites on the hormonal-activated Ca message system in the osteoblastic cell line UMR-106. The acute rise in [Ca2+]i induced by different calciotropic hormones [parathyroid hormone, prostaglandin E2 (PGE2)] was dose dependently blunted by 1,25-dihydroxyvitamin D [1,25(OH)2D3; half-maximal inhibitory concn approximately 5 x 10(-11) M] and was initially observed after 8 h of preincubation. The 1,25(OH)2D3 metabolite of vitamin D was two orders of magnitude more potent than 24,25(OH)2D3 and 25(OH)D3. To discern between an effect of 1,25(OH)2D3 on hormonal-induced Ca2+ entry through the plasma membrane channel vs. release of Ca2+ from internal stores, we suspended fura-2-loaded cells in Mn2+ rather than Ca2+ buffers. In cells preincubated with 1,25(OH)2D3, [Ca2+]i release (indicated by [Ca2+]i transient) was significantly blunted, whereas Mn2+ influx (indicating Ca2+ flux across the plasma membrane) was unaltered, suggesting a selective effect of 1,25(OH)2D3 on hormonal-activated release of Ca2+ from intracellular stores. 1,25(OH)2D3 also inhibited the PGE2-induced production of inositol 1,4,5-trisphosphate. We conclude that, in osteoblasts, chronic (hours) incubation with 1,25(OH)2D3 leads to attenuated stimulation of the [Ca2+]i transduction pathway by calciotropic hormones. This effect of 1,25(OH)2D3 may provide a cellular basis for the synergism between the effects of vitamin D and calciotropic hormones at the bone level.