Volume 8, Issue 2, Pages 303-316 (August 2001) The Retinoblastoma Protein Acts as a Transcriptional Coactivator Required for Osteogenic Differentiation  David M Thomas, Shannon A Carty, Denise M Piscopo, Jong-Seo Lee, Wen-Fang Wang, William C Forrester, Philip W Hinds  Molecular Cell  Volume 8, Issue 2, Pages 303-316 (August 2001) DOI: 10.1016/S1097-2765(01)00327-6

Figure 1 Expression of HPV16 E7 Attenuates Osteoblastic Differentiation of MC3T3-E1 Preosteoblasts (A) Cells were differentiated with ascorbic acid (AA). 50–100 μg of nuclear extracts (for E7, CBFA1, or pRb immunoblots) or total cell lysates for osteopontin (OP) were separated by SDS-PAGE and analyzed by immunoblotting. LXSN, cells stably expressing LXSN; E7, cells stably expressing HPV16 E7. (B) Differentiated (+AA) and undifferentiated (−AA) cells were analyzed for alkaline phosphatase activity. (C) Northern blot probed for mouse osteocalcin and GAPDH. (D) Medium was collected at the indicated time points following induction of differentiation. Osteocalcin levels were measured by radioimmunoassay. (E) After mineralization in the presence of β-glycerophosphate (BGP), the presence of calcified nodules was detected with Alizarin red-S. Calcified deposits stain red. Data in (B) and (D) are mean ± SEM from triplicate wells from a representative experiment of three total Molecular Cell 2001 8, 303-316DOI: (10.1016/S1097-2765(01)00327-6)

Figure 2 Primary Murine Embryonic Fibroblasts Derived from RB−/− Mice Fail to Undergo Full Osteoblast Differentiation in Response to BMP-2 (A) Alkaline phosphatase (ALP) activity in MEFs cultured in the presence or absence of BMP-2. Because of variation between embryo cultures, results are expressed as relative to untreated littermate control cultures. (B) RT-PCR for CBFA1 RNA in wild-type and RB−/− MEFs treated with or without BMP-2. Samples were diluted serially 1:2. (C) Medium was collected after culture in the presence of BMP-2. Osteocalcin levels were measured by radioimmunoassay. Results are expressed as relative to untreated littermate control cultures. Inset: RT-PCR analysis of osteocalcin RNA normalized with GAPDH. (D) Mineralization of MEFs treated with and without BMP-2 and stained with Alizarin red-S. Mineralization was similar in wild-type and p107+/−;p130+/− cultures. Data in (A) and (C) are mean ± SEM from triplicate wells from a representative experiment of two total Molecular Cell 2001 8, 303-316DOI: (10.1016/S1097-2765(01)00327-6)

Figure 3 pRb and CBFA1 Physically Interact In Vivo and In Vitro (A) Pull-downs using GST fusions to full-length CBFA1 and E7, and GST alone. 35S-labeled pRb, p107, and p130 were in vitro translated (IVT). 25% of GST-E7 pull-down and 100% of GST-CBFA1 or control was loaded. * indicates full-length pocket protein. (B) Binding of baculovirus-expressed, His-tagged pRb, or His-GST to 35S-methionine-labeled IVT CBFA1 and deletion mutants (described in [D]). (C) Binding of pRb and deletion mutants (described in [D]) expressed in Cos cells to GST-CBFA1 or GST. The blot was probed with anti-pRb (Ab-5, Calbiochem), except for del622–762 which is probed with 245. pRb and mutant proteins are indicated with arrows. (D) Schematic diagram of CBFA1, pRb, and deletion mutants used in IVT studies. Binding data are summarized at right. AD1–3, activation Molecular Cell 2001 8, 303-316DOI: (10.1016/S1097-2765(01)00327-6)

Figure 4 pRb Associates with CBFA1 on Osteoblast-Specific Promoters In Vitro and In Vivo (A) Colocalization of pRb and CBFA1-FLAG in a representative SAOS2 nucleus. Left panel is stained for pRb; middle panel is stained for FLAG; right panel, merged. (B) Chromatin immunoprecipitation (ChIP) was performed using differentiated MC3T3-E1 cells expressing LXSN or E7. Input: total DNA input for each sample; IP Rb, 245; IP CBFA1, C-19; Control IP, IP with an antibody matched for isotype (anti-FLAG M2 for pRb IP; anti-osteocalcin for CBFA1 IP); Unbound, PCR from the supernatant after IP; and Bound, PCR from IP. DNA input was equal in each PCR (10 ng). OC, osteocalcin; OP, osteopontin. Primer pairs and amplified sequences are described in Supplemental Data (see Supplemental Table at http://www.molecule.org/chi/content/full/8/2/303/DC1). (C) ChIP was performed using primary murine osteoblasts. The osteopontin and myogenin promoters were amplified as in (B) using the same primer pairs. (D) ChIPs from SAOS2 cells infected with adenovirus expressing vector control, pRb, CBFA1-FLAG, or both as indicated. Labels are as above. IPs were performed with anti-pRb or isotype matched control (anti-HA). Bound precipitated DNA was diluted serially as indicated and subjected to PCR for the proximal osteocalcin promoter sequence Molecular Cell 2001 8, 303-316DOI: (10.1016/S1097-2765(01)00327-6)

Figure 5 pRb Increases CBFA1-Dependent Transcription (A) Cell lines indicated were transfected with equivalent amounts of vector, CBFA1-FLAG, pRb, or both, together with the 6OSE2-luciferase reporter and CMV-β galactosidase. Samples were harvested after 48 hr and corrected for β-gal expression, and results expressed relative to control. The data are the mean ± SEM from triplicate wells of a representative experiment of three total. (B) SAOS2 cells were transfected with the constructs described together with the 6OSE2-luciferase reporter and CMV-β galactosidase, with the exception of lane 1 where 6OSE2-luc was replaced with a mutated 6OSE2 site (6OSE2mut) to correct for nonspecific activation. Results were normalized to 6OSE2mut (lane 1) and corrected for β-gal. The data are the mean ± SEM from triplicate wells as in (A). (C) SAOS2 cells were transfected with the constructs indicated, and samples were processed as in (A). The data are the means ± SEM of triplicate wells from five independent experiments, expressed as fold increase over control. (D) SAOS2 cells with a chromosomally integrated osteocalcin-luciferase reporter were infected with adenovirus vector, Ad-pRb, Ad-CBFA1-FLAG, or both. Cells were harvested at the indicated time points and analyzed for luciferase activity normalized to total protein content. Data are the mean ± SEM from triplicate wells expressed relative to control on day 1. SA-β-gal, senescence-associated β-galactosidase activity induced by pRb (+ = 10%–30% positive; +++ = >90% positive); G1 arrest, cell-cycle arrest induced by pRb (+++ = >90% G1). Bottom panel: Western blot of cell lysates from (D) blotted for pRb (245) and FLAG Molecular Cell 2001 8, 303-316DOI: (10.1016/S1097-2765(01)00327-6)

Figure 6 Binding and Transcriptional Activity of Naturally Occurring Tumor-Derived Mutants of pRb (A) Binding of pRb and tumor-derived mutants expressed in Cos cells to GST-CBFA1 or GST. The blot was probed with anti-pRb (Ab-5, Calbiochem). (B) SAOS2 cells were transfected with equivalent amounts of vector, CBFA1-FLAG, pRb, or both, together with the osteocalcin-firefly luciferase reporter and CMV-renilla luciferase. Samples were harvested after 48 hr and corrected for renilla expression, and results expressed relative to control. The data are the mean ± SEM from triplicate wells. This experiment was performed three times with similar results Molecular Cell 2001 8, 303-316DOI: (10.1016/S1097-2765(01)00327-6)

Figure 7 A Model of the Role of pRb in Osteogenic Differentiation and Terminal Cell Cycle Exit pRb is proposed to lead to differentiation and loss of proliferative capacity in osteoblasts both by repressing transcription through E2F and by activating transcription of differentiation-specific genes in collaboration with CBFA1. pRb also can augment p27KIP1 synthesis and increase sensitivity to other negative cell cycle regulators, but a direct role for CBFA1 in this process remains speculative Molecular Cell 2001 8, 303-316DOI: (10.1016/S1097-2765(01)00327-6)