Jane B. Lian

Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation can be examined in primary diploid cultures of fetal calvarial‐derived osteoblasts by the combination of molecular, biochemical, histochemical, and ultrastructural approaches. Modifications in gene expression define a developmental sequence that has 1) three principal periods: proliferation, extracellular matrix maturation, and mineralization; and 2) two restriction points to which the cells can progress but cannot pass without further signals. The first restriction point is when proliferation is down‐regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle and cell growth regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which an enhanced expression of alkaline phosphatase occurs immediately after the proliferative period, and later an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited; and 3) enhanced levels of expression of the osteoblast markers when collagen deposition is promoted, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and development of the osteoblast phenotype. The loss of stringent growth control in transformed osteoblasts and in osteosarcoma cells is accompanied by a deregulation of the tightly coupled relationship between proliferation and progressive expression of genes associated with bone cell differentiation.— Stein, G. S.; Lian, J. B.; Owen, T. A. Relationship of cell growth to the regulation of tissue‐specific gene expression during osteoblast differentiation. FASEB J. 4: 3111‐3123; 1990.

Concepts of osteoblast growth and differentiation: basis for modulation of bone cell development and tissue formation.

The combined application of molecular, biochemical, histochemical, and ultrastructural approaches has defined a temporal sequence of gene expression associated with development of the bone cell phenotype in primary osteoblast cultures. The peak levels of expressed genes reflect a developmental sequence of bone cell differentiation characterized by three principal periods: proliferation, extracellular matrix maturation and mineralization, and two restriction points to which the cells can progress but cannot pass without further signals. The regulation of cell growth and bone-specific gene expression has been examined during this developmental sequence and is discussed within the context of several unique concepts. These are (1) that oncogene expression in proliferating osteoblasts contributes to the suppression of genes expressed postproliferatively, (2) that hormone modulation of a gene is dependent upon the maturational state of the osteoblast, and (3) that chromatin structure and the presence of nucleosomes contribute to three-dimensional organization of gene promoters that support synergistic and/or antagonistic activities of physiologic mediators of bone cell growth and differentiation.

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. J. Cell. Biochem. 66:1–8, 1997.

Biological Functions of miR-29b Contribute to Positive Regulation of Osteoblast Differentiation

Bone tissue arises from mesenchymal cells induced into the osteoblast lineage by essential transcription factors and signaling cascades. MicroRNAs regulate biological processes by binding to mRNA 3′-untranslated region (UTR) sequences to attenuate protein synthesis. Here we performed microRNA profiling and identified miRs that are up-regulated through stages of osteoblast differentiation. Among these are the miR-29, miR-let-7, and miR-26 families that target many collagens and extracellular matrix proteins. We find that miR-29b supports osteoblast differentiation through several mechanisms. miR-29b decreased and anti-miR-29b increased activity of COL1A1, COL5A3, and COL4A2 3′-UTR sequences in reporter assays, as well as endogenous gene expression. These results support a mechanism for regulating collagen protein accumulation during the mineralization stage when miR-29b reaches peak levels. We propose that this mechanism prevents fibrosis and facilitates mineral deposition. Our studies further demonstrate that miR-29b promotes osteogenesis by directly down-regulating known inhibitors of osteoblast differentiation, HDAC4, TGFβ3, ACVR2A, CTNNBIP1, and DUSP2 proteins through binding to target 3′-UTR sequences in their mRNAs. Thus, miR-29b is a key regulator of development of the osteoblast phenotype by targeting anti-osteogenic factors and modulating bone extracellular matrix proteins.

A microRNA signature for a BMP2-induced osteoblast lineage commitment program

Bone morphogenetic proteins (BMPs) are potent morphogens that activate transcriptional programs for lineage determination. How BMP induction of a phenotype is coordinated with microRNAs (miRNAs) that inhibit biological pathways to control cell differentiation, remains unknown. Here, we show by profiling miRNAs during BMP2 induced osteogenesis of C2C12 mesenchymal cells, that 22 of 25 miRNAs which significantly changed in response to BMP2 are down-regulated. These miRNAs are each predicted to target components of multiple osteogenic pathways. We characterize two representative miRNAs and show that miR-133 directly targets Runx2, an early BMP response gene essential for bone formation, and miR-135 targets Smad5, a key transducer of the BMP2 osteogenic signal, controlled through their 3′UTR sequences. Both miRNAs functionally inhibit differentiation of osteoprogenitors by attenuating Runx2 and Smad5 pathways that synergistically contribute to bone formation. Although miR-133 is known to promote MEF-2-dependent myogenesis, we have identified a second complementary function to inhibit Runx2-mediated osteogenesis. Our key finding is that BMP2 controls bone cell determination by inducing miRNAs that target muscle genes but mainly by down-regulating multiple miRNAs that constitute an osteogenic program, thereby releasing from inhibition pathway components required for cell lineage commitment. Thus, our studies establish a mechanism for BMP morphogens to selectively induce a tissue-specific phenotype and suppress alternative lineages.

Expression of differentiated function by mineralizing cultures of chicken osteoblasts

This report documents osteoblast differentiation in vitro, as demonstrated by the 50-100X increase of proteins which are known markers of the osteoblast phenotype. Collagen type I and osteocalcin synthesis and accumulation, alkaline phosphatase activity, and matrix calcification show similar temporal relationships that are analogous to those seen during in vivo bone development. Chicken embryonic osteoblast progenitor cells were selected by initial growth at low densities in minimal medium. Upon subcultivation into nutrient-enriched medium at higher cell densities, near homogeneous populations of osteoblasts were obtained as demonstrated by the greater than 80% enrichment of cells positive for alkaline phosphatase activity. A comparison was made between cells grown in the presence or absence of 10 mM beta-glycerolphosphate (beta-GPO4), a chemical stimulant of matrix calcification, as a function of time. Cultures treated with beta-GPO4 showed visible calcification at Day 12 when culture monolayers became confluent. By Day 30, numerous large foci of calcification were visible and a 20-fold increase in calcium (Ca) content was observed. In contrast, untreated cultures had only a 3-fold increase in Ca content with many smaller diffuse areas of calcification. DNA, RNA, and total protein levels were nearly identical between the two cultures, indicating that beta-GPO4 had no marked effect on either cell proliferation or transcriptional activity. The major collagen type produced by either culture was type I, with no detectable type III as determined by CNBr peptide mapping and delayed reduction analysis. Alkaline phosphatase activity showed a rapid approximately 50-fold induction by Day 18 and remained elevated in control cultures. However, cultures treated with beta-GPO4 demonstrated a rapid 80% decline of enzyme activity after 18 days. In contrast, total osteocalcin levels showed a 100-fold induction by Day 18 and remained elevated in both control and beta-GPO4-treated cultures throughout the time period examined. While the overall levels of osteocalcin were the same in beta-GPO4-treated and untreated cultures, 2- to 5-fold more osteocalcin was associated with the more mineralized matrices of the beta-GPO4-treated cultures. In order to confirm the association of osteocalcin with areas of mineralization, co-localization of mineral to osteocalcin and collagen was carried out by combining vital labeling with tetracycline and immunofluorescent staining with anti-osteocalcin and anti-collagen antibodies. Both collagen and osteocalcin showed strong localization with areas of mineralization.(ABSTRACT TRUNCATED AT 400 WORDS)

Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression

We present an overview of Runx involvement in regulatory mechanisms that are requisite for fidelity of bone cell growth and differentiation, as well as for skeletal homeostasis and the structural and functional integrity of skeletal tissue. Runx-mediated control is addressed from the perspective of support for biological parameters of skeletal gene expression. We review recent findings that are consistent with an active role for Runx proteins as scaffolds for integration, organization and combinatorial assembly of nucleic acids and regulatory factors within the three-dimensional context of nuclear architecture.

Networks and hubs for the transcriptional control of osteoblastogenesis

We present an overview of the concepts of tissue-specific transcriptional control mechanisms essential for development of the bone cell phenotype. BMP2 induced transcription factors constitute a network of activities and molecular switches for bone development and osteoblast differentiation. Among these regulators are Runx2 (Cbfa1/AML3), the principal osteogenic master gene for bone formation, as well as homeodomain proteins and osterix. Runx2 has multiple regulatory activities, including activation or repression of gene expression, and integration of biological signals from developmental cues, such as BMP/TGFβ, Wnt and Src signaling pathways. Runx2 provides a new paradigm for transcriptional control by functioning as a principal scaffolding protein in nuclear microenvironments to control gene expression in response to physiologic signals (growth factors, cytokines and hormones). The protein serves as a hub for the coordination of activities essential for the expansion and differentiation of osteogenic lineage cells through the formation of co-regulatory protein complexes organized in subnuclear domains. Mechanisms by which Runx2 supports commitment to osteogenesis and determines cell fate involve its retention on mitotic chromosomes. Disruption of a unique protein module, the subnuclear targeting signal of Runx2, has profound effects on osteoblast differentiation and metastasis of cancer cells in the bone microenvironment. Runx2 target genes include regulators of cell growth control, components of the bone extracellular matrix, angiogenesis, and signaling proteins for development of the osteoblast phenotype and bone turnover. The specificity of Runx2 regulatory activities provides a basis for novel therapeutic strategies to correct bone disorders.

Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase

Competency for self‐renewal of human embryonic stem (ES) cells is linked to pluripotency. However, there is a critical paucity of fundamental parameters of human ES cell division. In this study we show that human ES cells (H1 and H9; NIH‐designated WA01 and WA09) rapidly proliferate due to a very short overall cell cycle (15–16 h) compared to somatic cells (e.g., normal diploid IMR90 fibroblasts and NT‐2 teratocarcinoma cells). The human ES cell cycle maintains the four canonical cell cycle stages G1, S, G2, and M, but the duration of G1 is dramatically shortened. Bromodeoxyuridine (BrdU) incorporation and FACS analysis demonstrated that 65% of asynchronously growing human ES cells are in S phase. Immunofluorescence microscopy studies detecting BrdU labeled mitotic chromosomes, Ki67 domains, and p220NPAT containing Cajal bodies revealed that the durations of the S (∼8 h), G2 (∼4 h), and M phases (∼1 h) are similar in ES and somatic cells. We determined that human ES cells remain viable after synchronization with either nocodazole or the anti‐tumor drug Paclitaxel (taxol) and have an abbreviated G1 phase of only 2.5–3 h that is significantly shorter than in somatic cells. Molecular analyses using quantitative RT‐PCR demonstrate that human ES cells and somatic cells express similar cell cycle markers. However, among cyclins and cyclin‐dependent kinases (CDKs), we observed high mRNA levels for the G1‐related CDK4 and cyclin D2 genes. We conclude that human ES cells exhibit unique G1 cell cycle kinetics and use CDK4/cyclin D2 related mechanisms to attain competency for DNA replication. J. Cell. Physiol. 209: 883–893, 2006.

Osteocalcin: isolation, characterization, and detection.

Publisher Summary This chapter discusses the isolation, characterization, and detection of osteocalcin. Osteocalcin is a small protein (5800 daltons) comprising 10-20% of the noncollagenous protein in the bone. Because osteocalcin is tightly adsorbed to the hydroxyapatite mineral phase of the bone, thorough extraction is best achieved by dissolving the mineral of finely pulverized bone. Hydroxyapatite is soluble in neutral 0.5M ethylenediaminetetraacetic acid (EDTA), as well as in a variety of mineral and organic acids. Uniformly high yields of 1-2 mg of osteocalcin per gram of dry bone are obtained with EDTA, whereas acid procedures often leave 10-25% of the protein behind, presumably because of precipitation during excursions through the osteocalcin isoelectric point. The use of radioimmunoassay for the measurement of osteocalcin offers the advantages of specificity, sensitivity, and technical simplicity. The method can easily detect nanogram quantities of the protein in bone extracts, cell cultures, and serum. The assay is based on the competition of the radioactively labeled antigen and an identical nonlabeled antigen for binding to a specific antibody. The amount of labeled antigen bound to the antibody is inversely proportional to the amount of unlabeled antigen present in the system.