Ribosomal protein L29/HIP deficiency delays osteogenesis and increases fragility of adult bone in mice

Abstract

Mice lacking HIP/RPL29, a ribosomal modulator of protein synthesis rate, display a short stature phenotype. To understand the contribution of HIP/RPL29 to bone formation and adult whole bone mechanical properties, we examined both developing and adult bone in our knockout mice. Results indicated that bone shortening in HIP/RPL29-null mice is due to delayed entry of chondro-osteoprogenitors into the cell cycle. Structural properties of adult null bones were analyzed by micro-computed tomography. Interestingly, partial preservation of cortical thickness was observed in null males indicating a gender-specific effect of the genotype on cortical bone parameters. Null males, and to a lower extent null females, displayed increased bone material toughness to counteract decreased bone size. This elevation in a bone material property was associated with increased bone mineral density only in null males. Neither male nor female null animals could withstand the same maximum load as gender-matched controls in three-point bending tests, and smaller post-yield displacements (and thus increased bone brittleness) were found for null animals. These results suggest that HIP/RPL29-deficient mice exhibit increased bone fragility due to altered matrix protein synthesis rates as a consequence of ribosomal insufficiency. Thus, sub-efficient protein translation increased fracture risk in HIP/RPL29-null animals. Taken together, these studies provide strong genetic evidence that the ability to regulate and amplify protein synthesis rates, including those proteins that regulate the cell cycle entry during skeletal development, are important determinants for establishment of normal bone mass and quality.

Figure 1

Cell density is increased in the reserve zone of HIP/RPL29 null epiphyses of distal embryonic femurs. Although the epiphysis/growth plates of d18.5 embryos (B) were shorter (white arrows) compared with controls (A), no overall alteration of cartilage organization was observed. However, the layer of proliferating (P) chondrocytes was shortened, and a significant increase in cellular density was seen in the reserve zone (R) of null embryos (C). BrdU-labeling revealed a small but significant decrease in chondroprogenitor proliferation in the R zone of developing nulls bones compared to controls (D). *** and * denote statistical significance differences (p < 0.001 and p < 0.05, respectively).

Figure 2

Immunostaining for early and late markers of chondrocyte differentiation on consecutive sections: type II (A, B) and type X collagen (C, D). Arrows (C, D) delineate the length of hypertrophic zones. Arrowheads in A indicate the periphery of developing bone.

Figure 3

Individual bone measurements showed a 30% decrease in wet bone weight (B) in HIP/RPL29 null (−/−). Also, overall bone length 7% less in HIP/RPL29-deficient bones (A). Representative mid-diaphyseal cross-sections showed a decrease in bone area of mutant animals compared to gender-matched controls (C). Cortical thickness was maintained in null males relative to controls. *** and * denote significant differences (p < 0.001 and p < 0.05, respectively).

Figure 4

EI, ultimate force, and PYD of HIP/RPL29-deficient and control femurs (A–C). BMD (D) measurement I_ML were obtained from μCT. The elastic modulus (F) was calculated from EI and I_ML. *, p < 0.05; n.s., non-significant. Data are mean ±std dev.