PMC

Model for the reciprocal regulatory relationship between osteoblast proliferation and Runx2 control of gene expression. Runx2 levels are modulated during the cell cycle and are maximal in G₁. We propose that Runx2 controls responsiveness of osteoblasts to mitogenic cues by altering the levels of components for multiple G protein-coupled receptor signaling pathways. In one of the pathways that is depicted here, Runx2 activates expression of Gpr30 and represses Rgs2. The net effect of these modulations is sensitization of cAMP/PKA signaling.

Overexpression of Rgs2 changes cell cycle distribution in MC3T3 osteoblasts. MC3T3 cells were transfected on coverslips with cytomegalovirus-driven expression vectors for Rgs2 or EGFP as control and fixed 36 h after transfection. Cells were examined by fluorescence microscopy for Ki67 and Rgs2 or GFP to assess the fraction of transfected cells in different cell cycle stages. The number of cells in each phase of the cell cycle was counted separately for both Rgs2 and EGFP-positive cell populations. A, representative micrographs of Ki67 immunofluorescence signals in MC3T3 cells. Similar to human Ki67, mouse Ki67 exhibits a cell cycle-related staining pattern that is related in part to dynamic changes in the organization of nucleoli at different stages of cell cycle. B, representative immunofluorescence image of MC3T3 cells positive for expression of Rgs2 or GFP. C, cell cycle distribution of MC3T3 cells expressing exogenous Rgs2 or GFP. We analyzed 100 cells each for three distinct coverslips (total n = 300), and error bars represent the range of these triplicate cell counts. Statistical significance of the data was determined by Student's t test, and values with p < 0.01 are indicated by an asterisk.

Rgs2 deficiency changes cell cycle distribution in Runx2 null osteoprogenitor cells. Runx2 null cells were transfected with 50 mm each of siRNA to Rgs2 or a nonsilencing (NS) siRNA control. Cells were re-plated 24 h after transfection in a 1:3 ratio and 12 h after replating treated with 1 μm PMA, 10 μm forskolin, or DMSO (vehicle control). After 24 h of treatment, cells were collected, and cell cycle distribution was analyzed by propidium iodide staining and FACS analysis. A, level of Rgs2 knockdown was monitored by qRT-PCR. Error bars represent S.E. between two different samples. The efficiency of siRNA transfection was monitored using a rhodamine-labeled control siRNA to GFP. Statistical significance of the data was determined by Student's t test, and values with p < 0.05 are indicated by asterisks. B, cell cycle distribution of Runx2 null cells treated with either siRNA for Rgs2 or nonsilencing RNA in the presence or absence of PMA (1 μm) or forskolin (10 μm) was established by FACS analysis. C, quantitation of the percentage of cells in S phase based on FACS data. Statistical significance of the data was determined by Student's t test, and values with p < 0.05 are indicated by asterisks, and values with p < 0.01 have two asterisks.

Runx2 growth control in Runx2 null osteoblasts progenitors requires C-terminal transcriptional activities and DNA binding of Runx2. Immortalized Runx2 null cells were infected with adenoviral vectors expressing wild type (WT) or mutant Runx2 (plus GFP), or GFP alone at comparable efficiencies of infection as described in . Growth curves were obtained by cell counting at daily intervals until 4 days after infection. Day -1 is the day of plating and day 0 is the day of infection. Error bars reflect variation observed in duplicate or triplicate independent experiments. Left, Runx2 null cells expressing wild type Runx2 (closed circles) grow slower than cells transfected with the corresponding empty vector (open circles). Middle, expression of Runx2 C-terminal point mutants (Yap interaction mutant Y433A (closed diamonds) and Smad/nuclear matrix interaction mutant HTY426–428AAA (dashed crosses)) results in similar growth inhibition as observed for wild type Runx2. A point mutation that abrogates Runx2 DNA binding activity (R197Q) (open diamonds) abrogates the growth inhibitory potential of Runx2. Growth curves for wild type Runx2 (dashed gray line) and empty vector (dashed black line) were taken from left panel and are shown for comparison. Right, expression of the C-terminal truncated Runx2 protein Δ361 (open triangles) does not inhibit growth, whereas Δ432 (closed triangles) exhibits a moderate growth inhibition phenotype compared with wild type Runx2 and empty vector (dashed lines taken from left panel).

Runx2 controls osteoblast proliferation by modulating the expression of several groups of related genes. Genes dependent on the growth regulatory C-terminal region of Runx2 were obtained by comparing Affymetrix cDNA expression microarrays. A, representative Runx2 target genes are shown that exhibit a consistent modulation on both day 1 and day 2 in cells expressing wild type Runx2 versus Runx2 Δ361 mutant or GFP (i.e. control vector). The genes selected here have a greater than 2.4-fold increase or decrease in expression (p < 0.05) on either day 1 or 2 (except Gpr54, which was selected as a member of the Gpr family of genes). Comparison of Runx2 wild type (WT) versus Runx2 Δ361 or wild type versus GFP yielded very similar changes in the expression of specific genes. Among the top genes identified in our list are classical bone-specific targets of Runx2 (e.g. osteopontin (Spp1), osteocalcin (Bglap2), and matrix metalloproteinase 13 (MMP13)). B, functional annotation clustering of 254 genes (>2-fold modulation with wild type Runx2 relative to Runx2 Δ361 at Day 1; p > 0.05) was performed using David 2.0 (data base for annotation, visualization, and integrated discovery, available on line). The pie chart shows the classification of annotated genes into different biological and cellular categories. C, Runx2-dependent genes related to G protein-coupled receptors encode proteins that operate at different steps within a signaling cascade.

GPR30 regulates proliferation of MC3T3 osteoblasts. MC3T3 cells were transfected with siRNAs (50 nm each) for Gpr30, Gpr54, Rgs4, or nonsilencing (NS) siRNA control in semi-confluent cultures and replated in a 1:3 ratio 24 h after transfection. Following a 36-h culture period, the subconfluent cell population was trypsinized, and cells were counted and harvested for RNA analysis. A, efficacy of each siRNA to knock down target gene expression was estimated by qPCR analysis. Levels of mRNA were plotted relative to RNA samples from untransfected control cells. Error bars represent standard error between two different RNA samples. Statistical significance of the data was determined by Student's t test, and values with p < 0.05 are indicated by one asterisk, and values with p < 0.01 have two asterisks. The efficiency of siRNA transfection was monitored using rhodamine-labeled control siRNA signal. B, biological effects of distinct siRNAs on proliferation of MC3T3 osteoblasts were determined by cell counting at 60 h after initiating siRNA treatment. Error bars represent the range of cell counts from triplicate samples. Statistical significance of the data was determined by Student's t test, and values with p < 0.01 are indicated by an asterisk. C, growth curves were obtained by cell counting at daily intervals until 4 days after treatment with nonsilencing (NS) RNA or siRNAs against Rgs4 or Gpr30 (#A, Ambion; #B, Dharmacon). Day 0 is the day when siRNA-transfected cells were replated. Error bars reflect variation observed in at least three independent measurements.

Expression of G protein-coupled receptor signaling components at distinct biological stages of osteogenic differentiation. A, expression of Runx2-dependent genes related to G protein-coupled receptor signaling and other markers was examined by qRT-PCR in mesenchymal progenitor cells obtained from the calvarial regions of Runx2 wild type and Runx2 null (KO) mice at embryonic day 17.5 and maintained in subconfluent culture for three passages. Several housekeeping genes were used as internal controls to validate differences in mRNA levels (Gapdh, Mcox, rRNA, and Hprt). mRNA levels of all different genes were plotted as fold change relative to each other (Gpr64 expression in Runx2 KO cells was arbitrary set as 1). Error bars represent standard error between four different mRNA preparations, each derived from multiple mouse embryos. Statistical significance of the data was determined by Student's t test, and values with p < 0.05 are indicated by one asterisk, and values with p < 0.01 have two asterisks. B, expression of representative GPCR-related genes, as well as Runx2 and bone phenotypic genes, was monitored by qRT-PCR analysis during growth of MC3T3 osteoblasts. MC3T3 cells were grown in proliferating media for 4 days until confluency. The levels for each mRNA were plotted relative to day 1 and normalized by 18 S rRNA as internal control. Error bars represent mean ± S.E. between three independent plates.

Validation of Runx2-dependent regulation of G protein-coupled receptor signaling components in osteoprogenitors and MC3T3 osteoblasts. Expression of Runx2-dependent genes related to G protein-coupled receptor signaling as determined by Affymetrix microarrays (A) using RNA samples obtained from reconstitution assays with Runx2 null cells (see Figs. and ) were validated using qRT-PCR analysis (B). The mRNA expression levels of cells infected with wild type Runx2 were plotted as fold change relative to cells infected with a vector expressing the Runx2 Δ361 mutant or GFP. Error bars reflect standard deviation of Affymetrix values from duplicate independent experiments and qRT-PCR values from triplicate experiments. Gene expression values measured by qRT-PCR were normalized using GAPDH as internal control. Statistical significance of the data was determined by Student's t test, and values with p < 0.05 are indicated by an asterisk, and values with p < 0.01 have two asterisks. C, Runx2 null cells were fixed and stained for Gpr30 and Rgs2 immunofluorescence upon infection with adenovirus expressing wild type Runx2, Δ361 mutant, or GFP (control vector). Wild type Runx2 modestly reduces Rgs2 protein level in the nucleus, albeit that Rgs2 detection is evident at bright aggregates in the cytoplasm. DAPI, 4′,6-diamidino-2-phenylindole. D, forced expression of Runx2 upon adenoviral infection in MC3T3 cells modulates the endogenous mRNA levels of the indicated genes as determined by qRT-PCR at day 1 after infection. The mRNA levels of GPCR-related genes in cells expressing wild type Runx2 were plotted as fold change over GFP and no virus controls and normalized using GAPDH as internal control. Statistical significance of the data was determined by Student's t test, and values with p < 0.05 are indicated by one asterisk, and values with p < 0.01 have two asterisks.

Runx2 reconstitution in osteoprogenitors from Runx2 null mouse calvaria. Cells were infected with adenoviral vectors expressing an IRES-driven GFP marker and either Runx2 wild type (WT) or different mutants defective for interactions with distinct regulatory cofactors. Virus expressing GFP alone was used as a control (GFP-EV). A, schematic representation of specific Runx2 mutations used in the context of functional protein domains of Runx2. Numbering is for the P1 isoform of Runx2 that starts with the amino acids MASNS and ends with the conserved residues VWRPY. Also indicated are a poly(Q/A) stretch, the runt homology domain (RHD) that controls DNA binding, C-terminal activation, and repression domains, as well as a nuclear matrix targeting signal. Micrographs were taken of transfected cells expressing an IRES-driven GFP at day 2 after infection to establish comparable infection efficiencies of each adenoviral vector (data not shown). B, Western blot analysis of exogenous Runx2 protein levels and different mutants shows comparable expression of Runx2 at day 1 after infection. The figure is a representative collage of comparable exposures of four distinct experiments with different sets of adenoviral vectors. Spliced lanes are indicated with dashed lines. C, expression of Runx2 mRNA levels in Runx2 null cells and induction of bone marker genes were monitored by qRT-PCR analysis. The mRNA levels for exogenous Runx2 were plotted relative to the average endogenous mRNA level in an equivalent amount of RNA from MC3T3 cells that was examined in parallel as external standard. RNA quantity was normalized relative to GAPDH as internal control. The mRNA levels of osteocalcin and osteopontin in control cells (no virus) are minimal, and values of 1 and below are close to background levels.