Amphiregulin lacks an essential role for the bone anabolic action of parathyroid hormone.

Although parathyroid hormone (PTH) has long been known to act as a bone anabolic agent when administered intermittently, the exact underlying mechanisms remain largely unknown. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, has been identified to be a PTH target gene in vitro and in vivo. Here, we used female global AREG knockout (AREG-KO) mice to explore the role of AREG in mediating the bone anabolic effects of PTH. AREG-KO mice were characterized by unchanged distal femoral cancellous bone mass and only subtle decreases in bone mineral density (BMD) and cortical thickness at the femoral midshaft at 3 and 8 months of age, relative to wildtype controls. AREG deficiency was associated with complex changes in the mRNA expression of other EGFR ligands in femoral cancellous bone osteoblasts in situ in 3-week-old mice. To examine the bone anabolic effects of PTH in the absence and presence of AREG, we injected 3-month-old AREG-KO females and wildtype control littermates with 80 µg/kg PTH or vehicle 5 times per week over 4 weeks. Intermittent PTH treatment of AREG-KO mice led to increases in femoral trabecular and cortical BMD, cortical thickness, endocortical and periosteal bone formation, cancellous bone formation rate, and serum osteocalcin, comparable to those observed in wildtype control mice. In conclusion, our data indicate that the bone anabolic effects of PTH do not require AREG, at least in 3-month-old female mice.

Osteoblast-specific overexpression of amphiregulin leads to transient increase in femoral cancellous bone mass in mice.

The epidermal growth factor receptor ligand amphiregulin (AREG) has been implicated in bone physiology and in bone anabolism mediated by intermittent parathyroid hormone treatment. However, the functions of AREG in bone have been only incipiently evaluated in vivo. Here, we generated transgenic mice overexpressing AREG specifically in osteoblasts (Col1-Areg). pQCT analysis of the femoral metaphysis revealed increased trabecular bone mass at 4, 8, and 10weeks of age in Col1-Areg mice compared to control littermates. However, the high bone mass phenotype was transient and disappeared in older animals. Micro-CT analysis of the secondary spongiosa confirmed increased trabecular bone volume and trabecular number in the distal femur of 4-week-old AREG-tg mice compared to control littermates. Furthermore, µ-CT analysis of the primary spongiosa revealed unaltered production of new bone trabeculae in distal femora of Col1-Areg mice. Histomorphometric analysis revealed a reduced number of osteoclasts in 4-week-old Col1-Areg mice, but not at later time points. Cancellous bone formation rate remained unchanged in Col1-Areg mice at all time points. In addition, bone mass and bone turnover in lumbar vertebral bodies were similar in Col1-Areg and control mice at all ages examined. Proliferation and differentiation of osteoblasts isolated from neonatal calvariae did not differ between Col1-Areg and control mice. Taken together, these data suggest that AREG overexpression in osteoblasts induces a transient high bone mass phenotype in the trabecular compartment of the appendicular skeleton by a growth-related, non-cell autonomous mechanism, leading to a positive bone balance with unchanged bone formation and lowered bone resorption.

A reporter mouse line with doxycyclin-inducible expression of ß-glucosidase.

Mouse lines allowing the inducible expression of transgenes became essential tools for studying gene function and for developing accurate animal models for human diseases. A key component of this tool is the availability of "reporter" lines, mice expressing transgenes encoding easily detectable enzymes or other proteins normally not associated with eukaryotic tissues. Such lines may be suitable for a number of applications, including lineage tracing, label-retaining experiments, and the identification and monitoring of regulatory elements important for tissue-specific gene expression. However, only a limited number of reporter lines suitable for inducible expression systems are available. Here, we employed pronuclear DNA microinjection to generate a new reporter mouse line that allows the inducible expression of ß-glucosidase, a recently reported stable and easily detectable protein, upon administration of doxycyclin to the drinking water. This novel line was established in the widely used inbreed background C57BL/6, and the transgene is transmitted between generations in a Mendelian fashion. When crossed to a K14-rtTA mouse line, activation of ß-glucosidase expression in the epidermal basal layer is easily detected in double-transgenic animals receiving doxycyclin, while no expression is seen in double-transgenic mice without doxycyclin treatment or in animals carrying only one transgene. We anticipate that this new mouse line will become a valuable tool for a number of applications in vivo, including label-retaining experiments and testing the appropriate regulation of rtTA cassettes under different promoters in novel transgenic mouse lines.