1. Rac signaling in osteoblastic cells is required for normal bone development but is dispensable for hematopoietic development
by Steven W. Lane, Serena De Vita, Kylie A. Alexander, Ruchan Karaman, Michael D. Milsom, Adrienne M. Dorrance, Amy Purdon, Leeann Louis, Mary L. Bouxsein, and David A. Williams
Blood
Volume 119(3):
January 19, 2012
©2012 by American Society of Hematology

2. Rac1 knock-down impairs growth and induces apoptosis in the osteoblast cell line OP9.
Rac1 knock-down impairs growth and induces apoptosis in the osteoblast cell line OP9. Photomicrograph of OP9 cells infected with nontargeting virus pLKO.1 (SCR; A) or infected with Rac1-shRNA 88 (pLKO.1 vector) demonstrating cytoskeletal elongation (B arrowheads). (C) Apoptosis (annexin V+ cells by flow cytometry) was induced by Rac1shRNA (88 and 92: apoptosis, 24.4% ± 2.6% and 17.4% ± 3.3%, respectively), but not with nontargeting hairpin (SCR, apoptosis 11.7% ± 0.2%, P < .01 vs sh88, P = .05 vs sh92). (D) Reduction in S-phase cell determined by bromodeoxyuridine incorporation caused by Rac1shRNA (88 and 92: S-phase 1.1% ± 0.9% and 1.9% ± 2.0%, respectively), but not nontargeting (SCR: S-phase 9.6% ± 4.0%, P < .05 for comparison with 88 and 92) control. (E) Representative image of bromodeoxyuridine incorporation in OP9 infected with nontargeting (SCR) control hairpin compared with Rac1shRNA 88 (F). All values are means ± SD. n = 3 per condition for each experiment.
Steven W. Lane et al. Blood 2012;119:
©2012 by American Society of Hematology

3. Rac deletion impairs osteoblast growth and differentiation in vitro.
Rac deletion impairs osteoblast growth and differentiation in vitro. Primary osteoblast cultures were transduced with an inducible Cre-expressing retrovirus (MSCV-CreER-puro) and treated with 4OHT or with EtOH as a control. (A) CFU-Fs were significantly reduced in Rac-deleted osteoblast cultures after induction of Cre expression by 4OHT (CFU-F Racdel 19 ± 4.4 EtOH vs 7.0 ± 1.6 4OHT), but not in WT controls. (B) CFU-Alk was reduced in Rac-deleted osteoblast cultures after 4OHT treatment (CFU-Alk Racdel 8.5 ± 1.7 EtOH vs 0.8 ± 0.5 4OHT). A modest reduction in CFU-Alk was observed with 4OHT treatment in WT osteoblast controls. (C) CFU-Alz was reduced in Rac-deleted osteoblast cultures after 4OHT treatment (CFU-Alz Racdel 8.5 ± 1.9 EtOH vs 2.5 ± 1.3 4OHT). (D) Apoptosis (annexin V+ cells by flow cytometry) was increased in WT and Rac-deficient osteoblast cultures after 4OHT treatment (Racdel 7.1% ± 2.7% EtOH vs 17.4% ± 6.1% 4OHT; WT 7.4% ± 0.5% EtOH vs 14.5% ± 2.0% 4OHT). All values are means ± SD. n = 3 per condition for each experiment.
Steven W. Lane et al. Blood 2012;119:
©2012 by American Society of Hematology

4. Osteoblast-restricted Rac deletion leads to defective bone acquisition in vivo.
Osteoblast-restricted Rac deletion leads to defective bone acquisition in vivo. Micro-CT images of distal femoral trabecular bone architecture demonstrating normal bone structure in Racdel mice (Cre− controls; A) and marked reduction of trabecular bone volume and abnormal architecture in Osx-Racdel mice (B). (C) Validation of complete excision of Rac alleles in osteoblasts isolated by flow cytometry in OsxRacdel mice but not Osx-WT controls. (D) Bone length was reduced in Osx-Racdel mice (10.7 ± 1.1 mm) compared with Cre− controls (13.4 ± 0.3 mm, P = .01). No difference was observed between Osx and WT controls (12.6 ± 0.5 mm vs 13.3 ± 0.8 mm, respectively, P = .14; n = 3-5 biologic replicates for each genotype). (E) Trabecular bone volume was reduced in Osx-Racdel mice (8.2% ± 2.6%) compared with Cre− controls (21.5% ± 4.4%, P < .01). No difference was observed between Osx and WT controls (13.9% ± 4.1% vs 19.1% ± 6.9%, respectively, P = .15; n = 4-6 biologic replicates for each genotype; F) Trabecular number was reduced in Osx-Racdel mice (3.5 ± 0.6/mm) compared with Cre− controls (5.7 ± 0.8/mm, P < .01). No difference was observed between Osx and WT controls (4.4 ± 0.9/mm vs 5.0 ± 1.0/mm, respectively, P = .15; n = 4-6 biologic replicates for each genotype; G) Cortical bone thickness was reduced in Osx-Racdel mice (0.078 ± mm) compared with Cre− controls (0.124 ± mm, P < .01). No difference was observed between Osx and WT controls (0.102 ± mm vs ± mm, respectively, P = .18). (n = 4-6 biologic replicates for each genotype; H.) Estimated cortical bone strength (as measured by the polar moment of inertia) was reduced in Osx-Racdel mice compared with Cre− controls (0.08 ± 0.03 mm4 vs 0.20 ± 0.05 mm4, P < .01). A trend to reduced cortical bone strength was observed in Osx transgenic controls compared with WT controls (0.15 ± 0.03 mm4 vs 0.22 ± 0.06 mm4, respectively, P = .06). (n = 4-6 biologic replicates for each genotype.) All values are means ± SD.
Steven W. Lane et al. Blood 2012;119:
©2012 by American Society of Hematology

5. Deletion of Rac in osteoblasts does not affect hematopoiesis.
Deletion of Rac in osteoblasts does not affect hematopoiesis. (A) Peripheral blood counts were similar between Racdel and Osx-Racdel mice for white blood cell count (WCC, 9.6 ± 2.2 × 103/μL vs 10.3 ± 3.4 × 103/μL, respectively, P = ns), hematocrit (HCT, 48.8% ± 3% vs 50.6% ± 0.8% respectively, P = ns) and platelet counts (973 ± 140 × 103/μL vs 1052 ± 164 × 103/μL, respectively, P = ns; n = 3-6 biologic replicates). (B) No difference in BM cellularity from Racdel and Osx-Racdel mice (9.6 ± 2.2 × 103/μL vs 10.3 ± 3.4 × 103/μL per 1 femur, respectively, P = ns), liver weight (1.3 ± 0.2 g vs 1.0 ± 0.1 g, respectively, P = ns), or spleen weight (0.06 ± 0.02 g vs 0.05 ± 0.01 g, respectively, P = ns; n = 3-6 biologic replicates). (C) Peripheral blood immunophenotype was similar between Racdel and Osx-Racdel for myeloid (10.8% ± 4.7% vs 13.5% ± 3.6%, respectively, P = ns), B cells (33.9% ± 17.8% vs 43.2 ± 5.6%, respectively, P = ns), and T cells (28.1% ± 28.1% vs 32.5% ± 7.3%, respectively, P = ns; n = 3-6 biologic replicates). (D) HSC and progenitor cell populations were similar between Racdel and Osx-Racdel mice; LKS ± 456 vs 2730 ± 303; multipotent progenitors (MPP), 1280 ± 323 vs 1580 ± 244; short-term HSCs (ST-HSC), 736 ± 127 vs 906 ± 131; and long-term HSCs (LT-HSC), 193 ± 29 vs 206 ± 52; P = ns for all, expressed per 1 × 106 BM cells, each dot represents individual biologic replicate. (E) Representative flow cytometry data plot demonstrating gating strategy for LKS+, MPP, ST-HSC, and LT-HSC. All values are means ± SD. Open boxes, Racdel; shaded boxes, Osx-Racdel.
Steven W. Lane et al. Blood 2012;119:
©2012 by American Society of Hematology

6. HSCs isolated from Osx-Racdel mice have normal function.
HSCs isolated from Osx-Racdel mice have normal function. (A) Osx-Racdel donor BM transplantation is able to sustain normal blood counts in lethally irradiated recipient mice (white blood cell count [WCC], 19.1 ± 7.9 × 103/μL; hematocrit [HCT], 47.6% ± 4.6%; platelet count, 616 ± 238 × 103/μL). n = 10 recipient mice. (B) Normal blood counts in lethally irradiated secondary recipient mice (WCC, 9.1 ± 2.0 × 103/μL; HCT, 46.6 ± 3.9%; platelet count, 880 ± 130 × 103/μL). (C) Similar BM competitive repopulation between Osx-Racdel and Cre− controls (donor chimerism, 37.4% ± 3.4% vs 35.1% ± 8.6% 16-week chimerism, P = ns; n = 6-7 recipients per condition). (D) Donor chimerism over time of BM derived from Osx-Racdel and Cre− controls (P = ns all time points). All values are means ± SD.
Steven W. Lane et al. Blood 2012;119:
©2012 by American Society of Hematology