Fig 1.
Phlpp1 cKOCtsk mice are not protected from ovariectomy-induced bone loss.
(A-F) Ovariectomy or Sham surgeries were performed on Phlpp1 cKOCtsk 12-week-old females and their wild-type littermates. Four weeks after surgery, the right femora were collected for analyses. (A) Experimental overview. Femora were collected, scanned via micro-CT and evaluated for (B) bone volume / total volume (BV/TV), (C) trabecular spacing (Tb. Sp.), (D) trabecular thickness (Tb. Th.), (E) trabecular number (Tb. N.), and (F) connective density (Conn D). *p<0.05.
Fig 2.
Phlpp1 cKOCtsk mice are not protected from ovariectomy-induced changes in osteoclast number.
(A, B) Ovariectomy surgeries were performed on Phlpp1 cKOCtsk 12-week-old females and their wild-type littermates. Four weeks after surgery, the right tibiae were collected for histomorphometric analyses. (A) Osteoclasts per bone perimeter (Ocl / B. Pm.), (B) osteoblasts per bone perimeter (Ob. Pm. / B. Pm.). Serum ELISAs for P1NP (C) and CTX-1 (D) were performed.
Table 1.
Percent change in bone parameters between control and Phlpp1 cKOCtsk mice following ovariectomy.
Fig 3.
Identification of ERE-containing genes that are differentially expressed by Phlpp1 cKOCtsk osteoclasts.
RNA from female day 4 Phlpp1 cKOCtsk osteoclasts or wild type counterparts was collected and used for high-throughput RNA sequencing. Listed genes were either (A) upregulated or (B) downregulated by two-fold or greater with an FDR < 0.05. (C) Venn diagram illustrating Phlpp1 cKOCtsk differentially regulated genes that also contain an ERE. (D) List of the top Phlpp1 cKOCtsk differentially expressed ERE-containing genes.
Fig 4.
Estrogen induces Phlpp1 expression, but represses Igfbp4 expression. (A) Osteoclasts progenitors were collected from 4-6-week-old Phlpp1 cKOCtsk females or their littermate controls. Western blotting was performed. (B) Osteoclast progenitors were collected from C57Bl/6 mice and cultured in the presence of the Phlpp inhibitor NSC 117079 (5μM) or vehicle control for 24 hours. Western blotting was performed. (C) Osteoclast progenitors were collected from control or Phlpp1-/- female mice were treated with 2 ng/ml estradiol (E2) for the indicated times and western blotting was performed. (D) Tibiae were collected from 12-week-old Phlpp1 cKOCtsk females of the control littermates. Immunohistochemical staining for Igfbp4 was performed, followed by counter staining with TRAP and fast green. (E) Tibiae were collected from 16-week-old Phlpp1 cKOCtsk females of the control littermates following ovariectomy. Immunohistochemical staining for Igfbp4 was performed, followed by counter staining with TRAP and fast green.
Fig 5.
Igfbp4 degradation attenuates enhanced osteoclast numbers induced by Phlpp1 deficiency in vitro.
(A, B) Osteoclasts were generated from 4-6-week-old female Phlpp1 cKOCtsk mice or their control littermates. Cultures were treated with 20 ng/ml rPAPP-A or PBS on days 0 and 3. (A) TRAP staining was performed and (B) the number of osteoclasts in each condition was evaluated. *p<0.05.