Activation of Nfatc2 in osteoblasts causes osteopenia

Abstract

Nuclear factor of activated T-cells (Nfat) c1 to c4 are transcription factors that play an undisputable role in osteoclastogenesis. However, Nfat function in osteoblastic cells is controversial. Constitutive activation of Nfatc1 and c2 in osteoblasts suppresses cell function, although the study of Nfat in vivo has yielded conflicting results. To establish the consequences of Nfatc2 activation in osteoblasts, we generated transgenic mice where a 3.6 kb fragment of the collagen type I α1 promoter directs expression of a constitutively active Nfatc2 mutant (Col3.6-Nfatc2). The skeletal phenotype of Col3.6-Nfatc2 mice of both sexes and of sex-matched littermate controls was investigated by microcomputed tomography and histomorphometry. Col3.6- Nfatc2 mice were born at the expected Mendelian ratio and appeared normal. Nfatc2 expression was confirmed in parietal bones from 1 and 3 month old transgenic mice. One month old Col3.6-Nfatc2 female mice exhibited cancellous bone compartment osteopenia secondary to a 30% reduction in bone formation. In contrast, cancellous femoral bone volume and bone formation were not altered in male transgenics, whereas osteoblast number was higher, suggesting incomplete osteoblast maturation. Indices of bone resorption were not affected in either sex. At 3 months of age, the skeletal phenotype evolved; and Col3.6-Nfatc2 male mice exhibited vertebral osteopenia, whereas femoral cancellous bone was not affected in either sex. Nfatc2 activation in osteoblasts had no impact on cortical bone structure. Nfatc2 activation inhibited alkaline phosphatase activity and mineralized nodule formation in bone marrow stromal cell cultures. In conclusion, Nfatc2 activation in osteoblasts inhibits bone formation and causes cancellous bone osteopenia.

Fig. 1. Design of the Col3.6-Nfatc2 transgene

A 3.6-kb fragment of the rat Col1a1 promoter cloned upstream a Kozak sequence (Kozak), and DNA coding for the human influenza hemagglutinin (HA), murine Nfatc2 harboring mutations that lead to the expression of a constitutively active protein (caNfatc2), and the bovine growth hormone polyadenylation signal (BGH pA).

Fig. 2. Col3.6-Nfatc2 transgenic mice overexpress Nfatc2 and have a normal general appearance

Male or female Col3.6-Nfatc2 transgenic mice (Nfatc2, black bars and filled circles) were compared to littermate wild type controls of the same sex (Control, white bars and open circles). In panel A, total RNA was extracted from parietal bones of 1 and 3 month old mice, and mRNA reverse-transcribed and amplified by qRT-PCR in the presence of specific primers. Data are expressed as Nfatc2 copy number, corrected for Gapdh copy number. Values are means ± SEM, n = 4. * Significantly different between Nfatc2 and Control, p < 0.05. Panel B, weight and femoral length of 1 and 3 month old mice. Values are means ± SEM, n = 4 – 9.

Fig. 3. One month old Col3.6-Nfatc2 transgenic female mice are osteopenic

Microcomputed tomography of proximal trabecular bone (top) and cortical bone at the mid-shaft (middle) of femurs, and toluidine blue staining of proximal femurs sections (bottom), from representative 1 month old male or female Col3.6-Nfatc2 transgenic mice (Nfatc2), and littermate wild type controls of the same sex (Control). Histological images were acquired at a 400× magnification, and image size was doubled digitally with Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA) after acquisition to achieve an 800× magnification. Red arrows indicate osteoblasts.

Fig. 4. Nfatc2 activation suppresses osteoblastogenesis in vitro

Bone marrow stromal cells were harvested from femurs of 1 month old Col3.6-Nfatc2 transgenic mice (Nfatc2, black bars), or littermate wild type controls (Control, white bars). Cells were pooled from mice of both sexes and cultured under conditions favoring osteoblastogenesis. In panel A, total RNA was extracted, and mRNA reverse-transcribed and amplified by qRT-PCR in the presence of specific primers. Data are expressed as Nfatc2 copy number corrected for Rpl38 copy number. Values are means ± SEM, n = 4. In panel B, alkaline phosphatase activity was determined and data are expressed as nanomoles of p-nitrophenol/min/μg of total protein. Values are means ± SEM, n = 6. In panel C, cultures were fixed with 4% formaldehyde in PBS and stained with alizarin red. Digital images of the cultures were acquired, and nodules counted with the analyze particles command of ImageJ software. Data are expressed as number of nodules/cm². Values are means ± SEM, n = 6. * Significantly different between Nfatc2 and Control, p < 0.05; ⁺ significantly different from Day 0 within Nfatc2 or Control cells, p < 0.05.