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. 2010 Jul 12;120(8):2755–2766. doi: 10.1172/JCI42106

Figure 3. OCL-targeted transgenic overexpression of ATF4 dramatically increases OCL differentiation and bone resorption and results in a severe osteopenic phenotype.

Figure 3

(A) Schematic representation of a transgene construct. An 1,846-bp fragment of the mouse Trap promoter was used to drive expression of full-length mouse ATF4 cDNA. Atf4 transgene expression in RANKL-differentiated and undifferentiated BMMs, BMSCs, or calvarial OBLs was measured by quantitative real-time RT-PCR using transgene-specific primers as described in Methods. (B–D) In vitro OCL differentiation. BMMs from 4-week-old WT and Trap-Atf4-tg mice (founder no. 2360) were differentiated into OCLs for 5 days followed by (B) Western blot analysis of ATF4, NFATc1, PU.1, CSFR1, and β-actin for loading; (C) real-time RT-PCR analysis for Trap, Cat K, Mmp9, Rank, Spi1, and Csfr1 mRNAs; and (D) TRAP staining of the BMM cultures. (E) TRAP staining. Tibial sections from 4-week-old WT and Trap-Atf4-tg mice were stained for TRAP activity. (F) TRAP+ OCLs (arrows) on trabecular surfaces of WT and Trap-Atf4-tg tibiae. Oc.S/BS and Oc.Nb/BPm values for primary and secondary spongiosa are shown in Table 2. (G) μCT analysis. Fixed nondemineralized femurs from 3-month-old male WT and Trap-Atf4-tg mice were used for μCT analysis as previously described (41). BV/TV, Tb.N, and Tb.Sp values are shown in Table 3. n = 3–7. *P < 0.01 versus WT. Original magnification, ×100 (D and E); ×200 (F).