Embryonic ablation of osteoblast Smad4 interrupts matrix synthesis in response to canonical wnt signaling and causes an osteogenesis imperfecta-like phenotype

Abstract

SummaryTo examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-b-BMP pathway, in Osx1 + cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of b-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt.

The p38α MAPK Function in Osteoprecursors Is Required for Bone Formation and Bone Homeostasis in Adult Mice

“…These data indicate that deletion of p38α in osteochondroprogenitors leads to a strong decrease in bone mass of both intramembranous and endochondral origins. They also corroborate previous reports showing that the bone phenotype in young Osx1-GFP::Cre mice was overcome by 12 weeks of age, with no differences observed between Osx1-GFP::Cre and control mice [20], [21]. There was no change in the number of osteocytes per area but, since the cortical area in p38α-deficient tibiae was lower, the total number of osteocytes was also lower in cortical samples from knockout mice (Fig.…”

Lrp5/β-Catenin Signaling Controls Lung Macrophage Differentiation and Inhibits Resolution of Fibrosis

“…The finding that Wnt/b-catenin signaling affects matrix processing is consistent with reports that link b-catenin signaling with the transforming growth factor-b pathway, fibroblast proliferation, and collagen synthesis (41)(42)(43)(44)(45). Among the top upregulated genes was lysyl oxidase, a collagen cross-linking enzyme that has been linked to disrupted matrix synthesis in a transforming growth factor-b-dependent manner (23,(46)(47)(48), although inhibition of another family member, LOXL2, protects from fibrosis (49) and is currently in phase 2 clinical trials for the treatment of idiopathic pulmonary fibrosis (50). Mmp-13, a macrophage-associated metalloproteinase, also known as collagenase-3, is also highly increased in Lrp5-null lungs after bleomycin injury, and has been shown to limit the development of fibrosis (25,26).…”

Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2

“…Fractured and nonfractured contralateral controls femurs were collected 5 days post fracture and 6 mm of mid diaphysis bone were dissected, cleaned of bone marrow, snap frozen in liquid nitrogen, and processed for total RNA extraction according using Trizol and RNeasy Plus kit, as previously described (Salazar et al, 2013).…”

“…Where indicated, cells were seeded to confluence and pretreated with cycloheximide (Sigma Aldrich, diluted to 25-100 mg/ml in growth medium) for 1 h prior to 4 h stimulation with Wnt3a+cyclohexamide. BMSCs were prepared from bone marrow from 4-to 6-month-old mice (male and female) collected as previously described (Salazar et al, 2013) and plated in BMSC medium [ascorbic acid-free α-MEM (Invitrogen) containing 20% FBS, 40 mM L-glutamine, 100 U/ml penicillin-G and 100 mg/ml streptomycin]. After 3 days, non-adherent cells were removed by vigorous washing.…”