The role of type XII collagen in bone formation 1Izu, Y; 2Zwolanek, D; 2Veit, G; 1Son, M; 2Koch, M; +1Birk, D E

+1University of South Florida, Tampa, FL, 2University of Cologne, Cologne, Germany dbirk@health.usf.edu

INTRODUCTION Type XII collagen is a members of the fibril associated collagens with interrupted triple helices (FACIT) subfamily. Type XII collagen exists as homotrimer of α1 chains and functions to regulate fibril spacing by bridging the fibrils. In addition, type XII collagen is a stress response molecule and is significantly expressed in bone compared to other mechanical response tissues such as tendon and skin [1]. In addition, murine osteoblastic MC3T3-E1 cells expressed type XII collagen with levels further elevated by mechanical strain [2]. However, the regulatory role of type XII collagen in bone formation in vivo is still undefined. To clarify the functional roles of type XII collagen in bone formation, we analyzed Col12a1 deficient mice, which were generated by homozygous recombination. MATERIALS AND METHODS Col12 a1 knockout mice were generated on mixed background with C57BL/6 and 129/SvJ. Homozygous recombination was carried out using a construct including neomycin(Neo)-poly adenylation signal (poly A) under the control of phosphoglycerol kinase (PGK) which was inserted into exon 2 to 5 of Col12a1 gene (Fig. 1). All animal studies

were performed in compliance with IACUC approved animal protocols. Fig. 1. A;Schematic representation of the wild type Col12a1 exons 1 to 8 and target locus. B; Southern blot analysis of genomic DNA of selected ES clones and their signal strength were measured by quantitative radiometry (C). D; PCR verification of the targeting events. Quantitative real-time PCR analysis was carried out using iCycler (Bio-Rad). Mineralized nodule analysis required bone marrow cells cultured in a standard growth medium containing 50 µg/ml ascorbic acid and 10 mM-glycerophosphate. After 21 days, cultures were stained with alizarin red and quantetated with the Image J analysis program. Histological analysis was performed using femurs, which were immersed in 4% paraformaldehyde at 4 °C overnight and decalcified in 15% EDTA. Cryosections were stained with H&E, Sirius Red for polarized microscopy analysis and immunofluorescence for type XII collagen. Osteocyte density was measured by randomly selecting 5 different cortical bone areas and counting eosteocyte number. Transmission electron microscopy was performed as described previously [4] Student’s t test was used to compare between wild type and Col12a1 knockout. The difference was judged to be statistically significant for p<0.05. All the numeral data in the results were presented as means S.D. RESULTS Type XII collagen was expressed in the periosteum and endosteum where osteoblasts actively secrete bone matrix localized using Immunofluorescence microscopy (Fig. 2). The expression of osteoblast differentiation markers, such as runx1, alp, ocn and col1a1, were examined based on real time PCR, suggesting that type XII collagen deficiency accelerated osteoblastic differentiation. However, mineralized nodule formation was decreased in bone marrow cells derived from Col12a1deficient mice. To clarify the in vivo phenotype of Col12a1 deficient mice, histological study was performed in cross sections of femur diaphyses. In contrast to the wild type, osteoblasts in Col12a1 deficient mice were hard to recognize and the border of individual cells were unclear in the periosteum (Fig. 3A). In bone matrix, osteocyte density was significantly increased in Col12a1 deficient mice (Fig. 3B) and where disorganized collagen arrangement was observed by polarized microscopy. Transmission electron microscopy revealed that while osteoblasts in the periosteum have abundant subcellular organelles

and are connected via processes in wild type mice, these structures were less developed in Col12a1 deficient mice (Fig. 4A,B). Such altered shape was observed not only in osteoblasts but also in osteocytes (Fig. 4C,D). Moreover, bone matrix in Col12a1 deficient mice showed disruption of normal collagen fibril arrangement. DISCUSSION In this study, we found that Col12a1 deficiency tended to accerelate osteoblast differentiation yet produce abnormal bone deposition associated with an altered shape of bone forming cells such as osteoblasts and osteocytes, suggesting that type XII collagen deficiency may regulate normal bone formation by maintaining the environment and communication among bone forming cells in the periosteum.

Fig. 2. Immunofluorescence localization of type XII collagen in cross sections of the femur (diaphysis) from P30 wild type mice. PO; periosteum, CB; cortical bone, BM; bone marrow, M; muscle, EO; endosteum (A) Bar = 250 µm. (B) Bar = 25 µm

Fig. 3. A;H&E staining of cross section of femurs (diaphysis) from P14 mice Arrows; osteoblasts, PO; periosteum, CB; cortical bone, BM; bone marrow, Bar = 50 µm. B; Osteocyte density in cortical bone matrix area was significantly increased in Col12a KO compared to WT at P14.

Fig. 4. Transition Electron Microscopy Image. Cross sections of the cortical bone area of WT (A, B) and Col12a1 KO ( C, D). Osteoblasts; white arrows, Osteocyte processes; yellow arrows, (A, C);Bar = 5 µm, (B, D);Bar = 1 µm, PO; periosteum, CB; cortical bone

ACKNOWLEDGEMENT This work was supported by NIH, NIAMS and US department of Health and Human Services. We thank Sheila Adams for help in Transmission electron microscopy analysis. REFERENCE [1] Arai et al., Cell Struct Funct. 2008. 33(2):203-210 [2] Oh et al., Dev Dyn. 1993. 196(1):37-46. [3] Ansorge et al., J Biol Chem. 2009. 284(13):8427-8438

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Paper No. 18 • 56th Annual Meeting of the Orthopaedic Research Society