Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype.

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.

Regulation of osteoblast gene expression and phenotype by polylactide-fatty acid surfaces.

Cell function is influenced by surface structure and molecules. Molecules that enhance cellular differentiation can be applied to tissue scaffold surfaces to stimulate endogenous tissue regeneration. The application of this approach to bone implants yields surfaces coated with factors (proteins, peptides, etc...) that promote the differentiation of osteoblasts, the cells that make bone. Increased bone formation leads to increased healing and union of the implant with endogenous bone. To obtain better control over surface coating we developed PLLA copolymers with allyl (PLLA-co-DAG) and 3-hydroxypropyl (PLLA-co-HP) side chains to which we can attach functional groups. Given the potential of fatty acids being able to incorporate into lipid bilayers and/or influence gene expression, we grafted different fatty acid side chains to PLLA-co-HP by esterifying the corresponding fatty acids with the PLLA-co-HP 3-hydroxypropyl side chains. The effects of the polymer modifications on osteoblasts were then evaluated. While cellular morphology differed between surface coatings, they did not reflect changes in cellular phenotype. Changes in gene expression were most evident with arachidonate and 3-hydroxypropyl side-chains which exhibited osteoblast differentiating capabilities. Linoleate, myristate, oleate, and stearate ester side-chains did not have a significant influence on osteoblast phenotype. Growth characteristics of osteoblasts did not differ between the fatty acid copolymer films, although cells grown on PLLA-co-HP exhibited a trend toward increased growth. Taken together our findings demonstrate that surface fatty acid composition can impact osteoblast phenotype.

Hepatocyte growth factor (HGF) adsorption kinetics and enhancement of osteoblast differentiation on hydroxyapatite surfaces.

Hepatocyte growth factor (HGF) is a growth factor that promotes angiogenesis (tissue vascularization), cell motility, and cell differentiation, making it a potentially beneficial coating for bone implants. However, very little is known about maximizing HGF attachment to surfaces of tissue-engineered scaffolds. Here, we examine methods and kinetics of HGF adsorption onto a dense hydroxyapatite (HA) surface (used in bone implants) and determine the influence of HGF coating on osteoblast phenotype/differentiation. We demonstrate that incubating HA with HGF in solution (and not allowing the solution to dry) resulted in maximal surface adsorption that was not enhanced by extending incubation time beyond 2 days. Daily shaking of the coated HA surface did not remove adsorbed HGF. To further examine the effect of HA on osteoblast phenotype, MC3T3-E1 preosteoblasts were seeded onto HA or HGF-HA surfaces. Gene expression analyses indicate that HGF coating enhanced osteoblast differentiation as demonstrated by increased runx2 (a transcription factor important for osteoblast lineage and differentiation), alkaline phosphatase (marker of mid stage differentiation) and osteocalcin (marker of late stage differentiation) mRNA levels. Taken together, our results demonstrate that HGF can serve as an excellent bone implant coating based on its ability to readily adsorb to HA surfaces, maintain integrity over time, and enhance osteoblast differentiation.

Electrostatic interactions as a predictor for osteoblast attachment to biomaterials.

The present study utilizes zeta (zeta)-potential analysis as an indicator of bonding of osteoblasts and whole bone to various biomaterials. Common metal alloys (316L stainless steel, CoCrMo, and Ti6Al4V) and bioceramics (hydroxyapatite and beta-tricalcium phosphate) used in orthopedic applications were suspended in particulate form in physiologic saline, both as-received and supplemented with bovine serum albumin (BSA). Metal alloys were also treated with NaOH washing to study the effect of such a surface treatment on the zeta-potential. The NaOH wash was found to increase the zeta-potential for CoCrMo and Ti6Al4V, but there was a decrease in the magnitude of the zeta-potential for 316L stainless steel. When the metal alloy powders were suspended in BSA-supplemented physiologic saline, the zeta-potential as a function of pH increased, thereby increasing the electronegativity gap and increasing the propensity for bonding between each of the metal alloys and bone. This increase is likely due to matrix proteins in the BSA, which adsorb onto the metal alloy surfaces, promoting bone growth. With the addition of BSA to each bioceramic system, a uniform decrease in zeta-potential was observed. However, the electronegativity gap remained large in each case, maintaining the anticipation of bonding. zeta-Potential analysis is an effective predictor of biomaterial attraction to osteoblasts and bone, providing a useful in vitro method for predicting such interactions.

Hydroxyapatite accelerates differentiation and suppresses growth of MC3T3-E1 osteoblasts.

Hydroxyapatite describes both the natural mineral phase of bone as well as the widely used calcium-phosphate implant substitute. Given that hydroxyapatite is a major component of the in vivo surface with which osteoblasts interact, it is surprising that most studies examining the regulation of osteoblast growth and differentiation utilize plastic surfaces. Here we demonstrate that the phenotype of mouse MC3T3-E1 osteoblasts is significantly altered on hydroxyapatite compared with plastic surfaces. Specifically, alkaline phosphatase activity and messenger RNA levels, markers of early stages of osteoblast differentiation, are increased in osteoblasts cultured on hydroxyapatite. The precocious appearance of alkaline phosphatase activity on the hydroxyapatite surface suggests that osteoblast differentiation is activated earlier compared with plastic surfaces. Osteocalcin expression, a marker of late-stage differentiation, is also increased on hydroxyapatite and further demonstrates enhanced differentiation. Cell counts indicate that fewer osteoblasts are present on hydroxyapatite versus plastic surfaces 24 h after plating. Measurement of osteoblast attachment, apoptosis, and necrosis indicated no differences between surfaces. In contrast, the number of bromodeoxyuridine-incorporating cells was significantly decreased on hydroxyapatite compared with plastic surfaces. Taken together, our findings indicate that hydroxyapatite enhances osteoblast differentiation while also suppressing growth.

Parathyroid hormone stimulates fra-2 expression in osteoblastic cells in vitro and in vivo.

PTH and PTH-related protein (PTHrP) are key mediators of skeletal development and homeostasis through their activation of the PTH-1 receptor. Previous studies have found that several AP-1 family members are regulated by PTH, such as c-fos, fra-1, and c-jun. There are numerous genes in the bone microenvironment that contain AP-1 sites, and different Fos family members are reported to have opposing transcriptional activities at AP-1 sites. The purpose of this study was to identify the effects of PTH on expression of the AP-1 protein complex member, fra-2, to extend our understanding of transcriptional regulators of PTH action. PTH induction of fra-2 messenger RNA (mRNA) levels in MC3T3-E1 preosteoblastic cells was maximal with 0.1 microM PTH (1-34). The expression in vitro was greatest 1 h after treatment and was present with N-terminal PTH but not PTH (7-34) or (53-84). Cycloheximide treatment induced fra-2 expression, and actinomycin D inhibited basal and PTHrP-induced expression. AP-1 protein in nuclear extracts of MC3T3-E1 cells was increased with PTH treatment at 3 h and consisted of high levels of Fra-2 protein, as evidenced by a supershift in an electrophoretic mobility shift assay and Western blot analysis. Up-regulation of steady-state fra-2 mRNA was also noted in vivo, where injection of PTH (1-34) (20 microgram) resulted in a more-than-7-fold maximal increase in fra-2 mRNA expression in the calvaria of mice, after 1 h of treatment. These data add to the transcriptional mediators induced by PTH and suggest that the interplay of AP-1 family members will provide insight into regulatory pathways of PTH and PTHrP for their anabolic and catabolic actions in bone.

Extracellular glucose influences osteoblast differentiation and c-Jun expression.

Insulin dependent diabetes mellitus, marked by high blood glucose levels and no insulin secretion, is associated with decreased bone mass and increased fracture rates. Analysis of bone histology suggests that osteoblast phenotype and function are influenced by diabetes. To determine if elevated extracellular glucose levels could directly influence osteoblast phenotype we treated mouse osteoblasts, MC3T3-E1 cells, with 22 mM glucose and analyzed osteoblast gene expression. Collagen I mRNA levels significantly increased while osteocalcin mRNA levels decreased 24 h after the addition of glucose. Expression of other genes, actin, osteopontin, and histone H4, was unaffected. Effects on collagen I expression were seen as early as 1 h after treatment. c-Jun, an AP-1 transcription factor involved in the regulation of osteoblast gene expression and growth, was also modulated by glucose. Specifically, an increase in c-jun expression was found at 1 h and maintained for 24 h following glucose treatment. Treatment of osteoblasts with an equal concentration of mannitol completely mimicked glucose treatment effects on collagen I and c-jun expression, demonstrating that osmotic stress rather than glucose metabolism is responsible for the effects on osteoblast gene expression and phenotype. Additional studies using staurosporine and Ro-31-8220 demonstrate that protein kinase C is required for the glucose up regulation of collagen I and c-jun. Taken together, our results demonstrate that osteoblasts respond to increasing extracellular glucose concentration through an osmotic response pathway that is dependent upon protein kinase C activity and results in upregulation of c-jun and modulation of collagen I and osteocalcin expression.

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Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex.

The AML/CBFA family of runt homology domain (rhd) transcription factors regulates expression of mammalian genes of the hematopoietic lineage. AML1, AML2 and AML3 are the three AML genes identified to date which influence myeloid cell growth and differentiation. Recently AML-related proteins were identified in an osteoblast-specific promoter binding complex that functionally modulates bone-restricted transcription of the osteocalcin gene. In the present study we demonstrate that in primary rat osteoblasts AML-3 is the AML family member present in the osteoblast-specific complex. Antibody specific for AML-3 completely supershifts this complex, in contrast to antibodies with specificity for AML-1 or AML-2, AML-3 is present as a single 5.4 kb transcript in bone tissues. To establish the functional involvement of AML factors in osteoblast differentiation, we pursued antisense strategies to alter expression of rhd genes. Treatment of osteoblast cultures with rhd antisense oligonucleotides significantly decreased three parameters which are linked to differentiation of normal diploid osteoblasts: the representation of alkaline phosphatase-positive cells, osteocalcin production, and the formation of mineralized nodules. Our findings indicate that AML-3 is a key transcription factor in bone cells and that the activity of rhd proteins is required for completion of osteoblast differentiation.

Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation.

Developmental studies of oncogene expression implicate the Fos and Jun family of transcription factors in the regulation of bone growth and differentiation. Promoters of many developmentally regulated genes, including osteocalcin, a marker of osteoblast differentiation, contain AP-1 sites that bind Fos/Jun dimers. Here, we demonstrate that the selective expression of fos- and jun-related genes is functionally related to the stage of osteoblast growth and differentiation in vitro. During osteoblast proliferation, nuclear protein levels of all seven activating protein-1 (AP-1) members are maximal. Subsequently, during the period of extracellular matrix maturation, levels decline. In fully differentiated osteoblasts, Fra-2 and (to a lesser extent) Jun D are the principal AP-1 members detectable by Western blot analysis. AP-1 complex composition and binding activity also exhibit developmental changes. All Fos and Jun family members are involved in AP-1 complex formation in proliferating cells, whereas Fra-2 and Jun D predominate in AP-1 complexes in differentiated osteoblasts. Overexpression of Fos and Jun family members in ROS 17/2.8 cells markedly affects the expression of an osteocalcin promoter-chloramphenicol acetyltransferase construct. Coexpression of only one AP-1 pair, Fra-2 and Jun D, stimulated reporter expression, whereas coexpression of other AP-1 pairs down-regulated expression (i.e. c-jun and any Fos family member) or had no effect (i.e. Fra-1 and Jun B). Promoter deletion analyses indicate that these effects are site specific. Consequential effects of Fra-2 on osteoblast differentiation are further demonstrated by antisense studies in which osteoblast differentiation and the development of a bone tissue-like organization were suppressed. Consistent with recent findings suggesting that AP-1 complex composition can selectively regulate gene transcription, our findings demonstrate that differential expression of Fos and Jun family members could play a role in the developmental regulation of bone-specific gene expression and, as a result, may be functionally significant for osteoblast differentiation.

Bone tissue-specific transcription of the osteocalcin gene: role of an activator osteoblast-specific complex and suppressor hox proteins that bind the OC box.

Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.

Selective expression of fos- and jun-related genes during osteoblast proliferation and differentiation.

Developmental studies of oncogene expression and transgenic animal studies implicate c-fos and other fos and jun family members in the regulation of bone tissue formation. Therefore, to initiate experimental examination of the hypothesis that expression of fos- and jun-related genes is functionally coupled to modulation of gene expression which supports bone development, we determined levels of expression of the principle fos and jun family members during progressive differentiation of normal rat calvaria-derived osteoblasts within two contexts. First, cellular mRNA levels were quantitated under conditions where expression of serum-induced early response genes had returned to basal levels. Our findings demonstrate high levels of c-fos, c-jun, and jun B mRNA transcripts during the proliferative period of osteoblast development, while expression of fra-1 and fra-2 is enhanced during the differentiation period. jun D is constitutively expressed during the time course exhibiting only a 30% decline in levels postproliferatively, and fos B mRNA is undetectable by Northern blot analyses. Late in the developmental sequence, apoptosis is evident. At this time, fra-1 expression is completely downregulated, while c-fos, fra-2, c-jun, jun B, and jun D show a dramatic enhancement in expression. Second, we addressed differential expression of fos and jun family members in relation to serum responsiveness as a function of stages of phenotypic development. Proliferating cells exhibit a prolonged induction of fos and jun family members in response to serum. While in differentiated cells, which are refractory to growth stimulus even when exposed to fresh serum every 2 days, a spike in fos and jun expression is observed. Thus, our data demonstrate significant differences in basal and serum responsiveness of fos and jun family members over the course of osteoblast differentiation. These findings are consistent with multiple lines of evidence linking activity of these early response genes to regulation of cell growth and development of the bone tissue phenotype.

Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.

The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.

Expression of cell growth and bone phenotypic genes during the cell cycle of normal diploid osteoblasts and osteosarcoma cells.

Establishing regulatory mechanisms that mediate proliferation of osteoblasts while restricting expression of genes associated with mature bone cell phenotypic properties to post-proliferative cells is fundamental to understanding skeletal development. To gain insight into relationships between growth control and the developmental expression of genes during osteoblast differentiation, we have examined expression of three classes of genes during the cell cycle of normal diploid rat calvarial-derived osteoblasts and rat osteosarcoma cells (ROS 17/2.8): cell cycle and growth-related genes (e.g., histone), genes that encode major structural proteins (e.g., actin and vimentin), and genes related to the biosynthesis, organization, and mineralization of the bone extracellular matrix (e.g., alkaline phosphatase, collagen I, osteocalcin, and osteopontin). In normal diploid osteoblasts as well as in osteosarcoma cells we found that histone genes, required for cell progression, are selectively expressed during S phase. All other genes studied were constitutively expressed both at the transcriptional and posttranscriptional levels. Alkaline phosphatase, an integral membrane protein in both osteoblasts and osteosarcoma cells, exhibited only minimal changes in activity during the osteoblast and osteosarcoma cell cycles. Our findings clearly indicate that despite the loss of normal proliferation-differentiation interrelationships in osteosarcoma cells, cell cycle regulation or constitutive expression of growth and phenotypic genes is maintained.