Inducible expression of Runx2 results in multiorgan abnormalities in mice

Abstract

Runx2 is a transcription factor controlling skeletal development, and is also expressed in extraskeletal tissues where its function is not well understood. Existing Runx2 mutant and transgenic mouse models do not allow the necessary control of Runx2 expression to understand its functions in different tissues. We generated conditional, doxycyline-inducible, triple transgenic mice (CMV-Cre;ROSA26-neo(flox/+)-rtTA;Tet-O-Runx2) to investigate the effects of wide spread overexpression of Runx2. Osteoblasts isolated from CMV-Cre;ROSA26-neo(flox/+)-rtTA; Tet-O-Runx2 mice demonstrated a dose-dependent effect of doxycycline to stimulate Runx2 transgene expression. Doxycycline administration to CMV-Cre;ROSA26-neo(flox/+)-rtTA;Tet-O-Runx2 mice induced Runx2 transgene expression in all tissues tested, with the highest levels observed in kidney, ovary, and bone. Runx2 overexpression resulted in deceased body size and reduced viability. With regard to bone, Runx2 overexpressing mice paradoxically displayed profound osteopenia and diminished osteogenesis. Induced expression of Runx2 in extraskeletal tissues resulted in ectopic calcification and induction of the osteogenic program in a limited number of tissues, including lung and muscle. In addition, the triple transgenic mice showed evidence of a myeloproliferative disorder and an apparent inhibition of lymphocyte development. Thus, overexpression of Runx2 both within and outside of the skeleton can have diverse biological effects. Use of tissue specific Cre mice will allow this model to be used to conditionally and inducibly overexpress Runx2 in different tissues and provide a means to study the post-natal tissue- and cell context-dependent functions of Runx2.

Fig. 1

Schematic diagram of inducible Tet-O-Runx2 transgene construct by doxycycline. A 445 bp of Tet-O-promoter, consisting of seven direct repeats of Tet Operator sequences [(Tet-O)₇] and a minimal CMV promoter, followed by a 1,587 bp mouse Runx2-II cDNA and a 848 bp SV40 intron plus poly A⁺ site was subcloned pW1 plasmid and linearized by ApaLI restriction enzyme.

Fig. 2

Breeding strategy for CMV-Cre-mediated and doxycycline-induced overexpression Runx2 mouse model in a triple transgenic system. A triple Cre-loxP system was used to generate global inducible Tet-O-Runx2-II transgenic mice by conditional deletion of the floxed PGK-neo cassette in all tissues using CMV-Cre through two-round crossing Tet-O-Runx2-II, CMV-Cre, and ROSA26-neo^flox/flox-rtTA mice. For the first round, Tet-O-Runx2-II mice were crossed with CMV-Cre to generate CMV-Cre;Tet-O-Runx2-II mice. For the second round, CMV-Cre;Tet-O-Runx2-II mice were mated with ROSA26-neo^flox/flox-rtTA mice to generate CMV-Cre; ROSA26-neo^flox/+-rtTA;Tet-O-Runx2-II mice (triple transgenic mice).

Fig. 3

Gross appearance and Runx2 expression in Dox treated CMV-Cre; ROSA26-neo^flox/+-rtTA; Tet-O-Runx2-II (Triple Tg) and control mice. Dox (2 mg/ml in 5% sucrose solution) was added to the drinking water to 3-week-old mice for 2 weeks. A: Gross appearance of 5-week-old mice after treated with Dox for 2 weeks. B: Kaplan–Meier survival curve of CMV-Cre;ROSA26-neo^flox/+-rtTA;Tet-O-Runx2-II (Triple Tg) and control mice. Dox treatment was started at day 0 in 3-week-old mice. C: Runx2-II transgene expression in various tissues after Dox treatment of CMV-Cre; ROSA26-neo^flox/+-rtTA; Tet-O-Runx2-II mice by quantitative RT-PCR. Data represent the mean ± SD from four individual samples.

Fig. 4

Musculoskeletal phenotype in Dox treated CMV-Cre; ROSA26-neo^flox/+-rtTA; Tet-O-Runx2-II (Triple Tg) and control mice. A: Body weight. B–D: DEXA analysis of bone density (BMD), fat mass and lean body mass. Data represent the mean ± SD from a minimum of four samples. Asterisk (^*) indicates significant difference from control mice at P <0.05.

Fig. 5

Osteoblast-mediated bone formation and bone resorption in Dox-induced Runx2-II transgenic mice. A: μCT analysis of femoral trabecular and cortical bone; B: Bone mineral apposition rate (MAR) by bone histomorphometric analyses. C: The levels of serum TRAP. D: Osterix and Osteocalcin messages of tibias by real time RT-PCR. E: Inducible regulation of Runx2-II and its downstream gene expression [Osteocalcin, Bone sialoprotein (Bsp)] mRNA abundance by quantitative RT-PCR in cultured osteoblasts. Data represent the mean ± SD from a minimum of four samples. Asterisk (^*) indicates significant difference from control mice at P <0.05.

Fig. 6

Variable effects of overexpressing Runx2-II to stimulate an osteogenic program in different tissues in Dox treated CMV-Cre;ROSA26-neo^flox/+-rtTA;Tet-O-Runx2-II mice. A: Multiple organs calcification with Alizarin red staining. B: Quantification of Alizarin red staining. C–I: Expression of Runx2 downstream targeting genes such as Osterix and Osteocalcin messages by real time RT-PCR in lung, spleen, muscle, aorta, heart, liver, and kidney samples. Data represent the mean ± SD (n = 4). Asterisk (*) indicates significant difference from control mice at P <0.05.

Fig. 7

Hematological abnormalities in Dox treated CMV-Cre; ROSA26-neo^flox/+-rtTA; Tet-O-Runx2-II (Triple Tg) and control mice. Gross appearance, weight and H&E stained histological section of the spleen (A), thymus (B), and liver (C). D: Bone marrow smear (Wright-Giemsa stain) showing altered myeloid-erythroid ratio in Runx2 overexpressing mice (low power, upper panel) and uniform appearing immature “blast” like cells in Runx2-II expressing mice (high power, lower panel). Data represent the mean ± SD (n = 4). Asterisk (^*) indicates significant difference from control mice at P <0.05.

Fig. 8

Increase in neutrophil and myeloid and monocyte precursors (Mac1⁺⁻Gr1^int) in Dox-induced Runx2-II triple transgenic mice. A: Peripheral blood count (n = 3) showed that the percentage of neutrophil and lymphocyte was upside down in Runx2 transgenic (Triple Tg) mice compared with control mice treated with Dox for 1 week. B,C: Flow cytometry analyses of bone marrow (BM) cells from control and Runx2-II transgenic (Tg) mice treated with Dox for 1 week. The data showed that the percentage and absolute number of bone marrow myeloid and monocyte precursors (Mac1⁺Gr1^int) in Runx2 transgenic mice significantly increased when compared with control BM. Data are expressed as the mean ± SD (n = 3). Asterisk (^*) indicates significant difference from control mice at P <0.05.