HES1 (hairy and enhancer of split 1) is a determinant of bone mass

Abstract

HES1 (hairy and enhancer of split) is a transcription factor that regulates osteoblastogenesis in vitro. The skeletal effects of HES1 misexpression were studied. Transgenic mice where a 3.6-kilobase fragment of the collagen type 1 α1 promoter directs HES1 overexpression were created. Transgenics were osteopenic due to decreased osteoblast function in female and increased bone resorption in male mice. HES1 impaired osteoblastogenesis in vitro, and transgenic osteoblasts enhanced the resorptive activity of co-cultured osteoclast precursors. Mice homozygous for a Hes1 loxP-targeted allele were bred to transgenics, where the paired-related homeobox gene enhancer or the osteocalcin promoter direct Cre recombinase expression to inactivate Hes1 in the limb bud or in osteoblasts. To avoid genetic compensation, Hes1 was inactivated in the context of the global deletion of Hes3 and Hes5. Hes3 and Hes5 null mice had no skeletal phenotype. Hes1 inactivation in the limb bud increased femoral length and trabecular number. Hes1 inactivation in osteoblasts increased trabecular bone volume, number, and connectivity due to increased mineral apposition rate and suppressed bone resorption. Hes1 inactivation in vitro increased alkaline phosphatase expression and suppressed the resorptive activity of co-cultured osteoclast precursors. In conclusion, by inhibiting osteoblast function and inducing bone resorption, HES1 is an intracellular determinant of bone mass and structure.

FIGURE 1.

Skeletal phenotype of 3-month-old male (left) and female (right) heterozygous HES1 transgenic mice (black bars) and wild type littermate controls (white bars). In A, the weight (g), femoral length (mm), and total BMD (g cm⁻²) are shown. Values are means ± S.E., n = 6–8. *, Significantly different from control mice, p < 0.05. Shown in B and C are von Kossa staining (B; final magnification, 40×) and calcein/demeclocycline labeling (C; final magnification, 100×) of representative femoral sections. D, microcomputed tomography of representative femurs.

FIGURE 2.

Effects of HES1 overexpression on osteoblastic function in vitro. In A and B, calvarial osteoblasts (A) and bone marrow stromal cells (B) were harvested from HES1 transgenics (black bars) and wild type littermate controls (white bars). Total RNA was extracted at confluence and after 7 days of culture in conditions favoring osteoblastogenesis; mRNA was reverse-transcribed and amplified by real time RT-PCR in the presence of specific primers. Data are expressed as Hes1, alkaline phosphatase (Ap), and osteocalcin copy number, determined by real time RT-PCR, corrected for Rpl38 expression. Values are means ± S.E., n = 4. In C, calvarial osteoblasts harvested from HES1 transgenics (HES1) and wild type littermate controls (WT) were co-cultured with splenocytes harvested from wild type mice, in the presence of 10 nm 1,25-dihydroxyvitamin D₃ (Vit.D; black bars) or control vehicle (white bars). Data are expressed as % of resorbed area. Values are means ± S.E., n = 4–6. *, significantly different from wild type cells, p < 0.05. + significantly different from cells treated with vehicle, p < 0.05.

FIGURE 3.

Skeletal phenotype of 1-month-old male Prx1-Cre/+;Hes1^Δ/Δ;Hes3^−/−;Hes5^−/− conditional null mice (Hes1/3/5 null) and Hes1^loxP/loxP;Hes3^−/−;Hes5^−/− littermate controls (Control) of the same sex. In A, a representative PCR demonstrates the recombination of the Hes1^loxP allele in calvarial DNA extracts. In B, the weight (g), femoral length (mm), and total BMD (g cm⁻²) of Prx1-Cre/+;Hes1^Δ/Δ;Hes3^−/−;Hes5^−/− mice (black bars) and controls (white bars) are shown. Values are means ± S.E., n = 5–6. *, significantly different from control mice, p < 0.05. C and D, von Kossa staining (C; final magnification, 40×) and calcein/demeclocycline labeling (D; final magnification, 100×) of representative femoral sections. Shown in E are microcomputed tomography of representative femurs.

FIGURE 4.

Skeletal phenotype of 1 month (left), 3 month (middle) and 6 month (right) old male Oc-Cre/+;Hes1^Δ/Δ;Hes3^−/−;Hes5^−/−conditional null mice (Hes1/3/5 null) and Hes1^loxP/loxP;Hes3^−/−;Hes5^−/− littermate controls (Control) of the same sex. A, representative PCR demonstrates the recombination of the Hes1^loxP allele in calvarial DNA extracts. B, the weight (g), femoral length (mm), and total BMD (g cm⁻²) of Oc-Cre/+;Hes1^Δ/Δ;Hes3^−/−;Hes5^−/− mice (black bars) and controls (white bars). C and D, von Kossa staining (C; final magnification, 40×) and calcein/demeclocycline labeling (D; final magnification, 100×) of representative femoral sections. E, microcomputed tomography of representative femurs.

FIGURE 5.

Consequences of Hes1 inactivation on osteoblastic function in vitro. In panel A, Calvarial osteoblasts were harvested from Oc-Cre/+;Hes1^Δ/Δ;Hes3^−/−;Hes5^−/− conditional null mice (black bars) and Hes1^loxP/loxP;Hes3^−/−;Hes5^−/− littermate controls (white bars). Total RNA was extracted at confluence and after 7 days of culture in conditions favoring osteoblastogenesis; mRNA was reverse-transcribed and amplified by real time RT-PCR in the presence of specific primers. Data are expressed as Hes1, alkaline phosphatase (Ap), and osteocalcin copy number, determined by real time RT-PCR, corrected for Rpl38 expression. Values are means ± S.E., n = 4. *, significantly different from control cells, p < 0.05. In B, calvarial osteoblasts harvested from Oc-Cre/+;Hes1^Δ/Δ;Hes3^−/−;Hes5^−/− conditional null mice (Hes1/3/5 null) and Hes1^loxP/loxP;Hes3^−/−;Hes5^−/− littermate controls (Control) were co-cultured with splenocytes harvested from wild type mice, in the presence of 10 nm 1,25-dihydroxyvitamin D₃ (Vit.D, black bars) or control vehicle (white bars). Data are expressed as % of resorbed area. Values are means ± S.E., n = 4–6. *, significantly different from control cells, p < 0.05. +, significantly different from cells treated with vehicle, p < 0.05.