Abstract
Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.
Highlights
Site-1 protease (S1P3; known as the membrane-bound transcription factor protease, site-1) is a proprotein convertase
Our study shows that the removal of S1P activity in postnatal mice results in loss of chondrocyte hypertrophic differentiation in parallel with Col II retention in the cell and the eventual loss of the growth plate through apoptosis
In situ hybridization (ISH) analyses demonstrated the loss of S1P in the cartilage of both E10.5–E18.5 and E13.5–E18.5 S1Pcko-ER(T) mice indicating that the use of Col2CreERT system successfully eliminated S1P expression and that the resulting phenotype is due to loss of S1P activity
Summary
Generation of S1Pcko-ER(T) Mice—To generate S1Pcko-ER(T) mice, S1Pf/f mice (mice homozygous for the S1Pflox allele, where S1P exon 2 is floxed [10]) were bred with Col2CreERT mice (mice in which the Cre recombinase is directed by the cartilagespecific Col2a1 promoter limiting its expression primarily to cartilage and is tamoxifen-dependent requiring tamoxifen injections for Cre recombinase activity [9]) to produce S1Pf/ϩ; Col2CreERT mice (mice heterozygous for the S1Pflox allele and positive for the Col2-CreERT transgene). Pregnant female mice from crosses of S1Pf/f and S1Pf/f;Col2CreERT mice were given a single dose of 3 mg of tamoxifen at 10.5 or 13.5 days postcoitus and harvested at embryonic (E) stage 18.5 to give E10.5–E18.5 and E13.5–E18.5 S1Pcko-ER(T) mice, respectively. Newborn mouse pups (P1), both S1Pf/f (WT) and S1Pf/f;Col2CreERT, were injected with 1 mg of tamoxifen suspension in corn oil and harvested when 3, 5, 7, or 14 days old and are referred to as P1–P3, P1–P5, P1–P7, or P1–P14 WT and S1Pcko-ER(T) mice, respectively. Morphological and Histological Analyses—Histological analyses by Safranin O, Fast Green, and hematoxylin staining were done on formalin-fixed, paraffin-embedded tissues cut 5 m thick. Visualized for incorporation of calcein (FITC filter) and alizarin (TRITC filter) by fluorescent microscopy (Olympus BX51), and images were digitized and merged (Olympus DP70)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
