Differential expression of both extracellular and intracellular proteins is involved in the lethal or nonlethal phenotypic variation of BrtlIV, a murine model for osteogenesis imperfecta

Abstract

This study used proteomic and transcriptomic techniques to understand the molecular basis of the phenotypic variability in the bone disorder osteogenesis imperfecta (OI). Calvarial bone mRNA expression was evaluated by microarray, real-time, and comparative RT-PCR and the bone proteome profile was analyzed by 2-DE, MS, and immunoblotting in the OI murine model BrtlIV, which has either a moderate or a lethal OI outcome. Differential expression analysis showed significant changes for eight proteins. The expression of the ER stress-related protein Gadd153 was increased in lethal mice, whereas expression of the chaperone alphaB crystallin was increased in nonlethal mice, suggesting that the intracellular machinery is involved in the modulation of the OI phenotype. Furthermore, in lethal BrtlIV, the increased expression of the cartilaginous proteins Prelp, Bmp6, and Bmp7 and the lower expression of the bone matrix proteins matrilin 4, microfibril-associated glycoprotein 2, and thrombospondin 3 revealed that both a delay in skeletal development and an alteration in extracellular matrix composition influence OI outcomes. Differentially expressed proteins identified in this model offer a starting point for elucidating the molecular basis of phenotypic variability, a characteristic common to many genetic disorders. The first reference 2-DE map for murine calvarial tissue is also reported.

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

“…42 We recently described ER stress in Brtl fibroblast and calvarial bone, as well as in MSCs differentiating toward the osteoblastic lineage. [30][31][32] In the latter case, we showed an increase in HSP47, a specific molecular chaperone known to bind folded type I procollagen molecules in the ER, similar to previous reports in some OI cases. 43 Thus, we evaluated the expression of HSP47 following silencing and found a significant reduction in F-Mut shRNA-transduced Brtl cells, compared with control fibroblasts, as might be expected based on about 40% total collagen suppression.…”

“…The expression level of the target gene was normalized to the housekeeping gene Gapdh (Mm99999915_g1, Applied Biosystems) and the relative expression was calculated using the DDCt method. The evaluation of the specific expression of each Col1a1 allele was then performed by means of allele-specific real-time PCR as described in Forlino et al 30 Collagen analysis F-Mut shRNA transduced and parental Brtl fibroblasts were plated (2.5 Â 10 5 /3 cm Petri dishes) and allowed to adhere for 24 h. Collagen synthesis was stimulated by incubating the cells for 24 h before protein recovery with 100 mg/ml sodium ascorbate in DMEM containing 1% FBS. Type I collagen was extracted and purified from media and cell layer as described previously.…”

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

“…In untreated animals from our colony, BrtlIV pups had a survival rate 2 times lower compared with WT and, at the same age of 2 months, 65.5% were WT and 34.5% were heterozygous BrtlIV, consistent with previous reports. 20,41 The significantly higher (P ϭ .04) proportion of surviving BrtlIV animals in transplanted mice indicated that the treatment rescued the genotype-related lethality in our murine model.…”

“…45,46 From the intensities of the ␣1(I)-S-S-␣1(I) and ␣1(I) bands, we estimated the fraction of collagen molecules with 2 mutant chains as 26.7% plus or minus 4.3% in trabecular and 25.7% plus or minus 2.5% in cortical bone from untreated animals, consistent with previous reports. 41,46 In treated animals, this fraction decreased with increasing bone marrow engraftment of donor cells ( Figure 4B). In animals with the highest engraftment (1.9% Ϯ 0.1%), this fraction was 20.4% plus or minus 2.9% in trabecular and 20.9% plus or minus 2.6% in cortical bone (P Ͻ .05; Figure 4 and supplemental Table 2).…”

“…The second dose was added after 3 to 4 days. After 1 week, the reaction was stopped with acetic acid (0.5 M final concentration), and collagen was precipitated by raising the NaCl concentration to approximately 1 M. The precipitated collagen was washed with 70% ethanol, resolubilized in 0.1 M Na-carbonate, 0.5 M NaCl, pH 9.3, fluorescently labeled with Cy5 (Crt Healthcare), 41 and separated on precast, gradient, Tris-acetate minigels (3%-8%; Invitrogen). The gels were scanned on an FLA5000 fluorescence scanner (Fuji Medical Systems) and analyzed with the ScienceLab software supplied by the manufacturer (Fuji Medical Systems).…”