Function Of Skeletal Matrix Genes

Dental & Craniofacial Research

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Abstract

The goal of the Section is to explore the function of small leucine-rich proteogycans in skeletal tissues. Our working hypothesis is that SLRPs have important roles in controlling the structure and function of bones and teeth. We have tested this hypothesis by creating mice deficient in one or more SLRPs and by examining the resulting phenotypes at the structural, cellular and molecular levels. [unreadable] [unreadable] BIGLYCAN DEFICIENCY INCREASES OSTEOCLAST DIFFERENTIATION AND ACTIVITY DUE TO DEFECTIVE OSTEOBLASTS (50%)[unreadable] Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts is regulated by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. We now show that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts and their precursors using both in vivo and in vitro experiments. The in vivo osteolysis experiment showed that lipopolysaccharide (LPS) induced osteolysis occurred more rapidly and extensively in biglycan deficient mice compared to wildtype mice. To further understand the mechanism of action, we determined the effects of biglycan on 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3)-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate-resistant acid phosphatase-positive multinuclear cells (TRAP+ MNCs) appeared earlier and more extensively in the co-cultures containing calvarial cells from biglycan deficient mice than wildtype mice, regardless of the genotype of osteoclast precursors. The osteoblast abnormality that stimulated osteoclast formation appeared to be independent of the differential production of soluble (Receptor Activator for Nuclear Factor Kappa B Ligand) RANKL and (Osteoprotegerin) OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in SLPI (secretory leukocyte protease inhibitor) in biglycan deficient osteoblasts treated with 1, 25-(OH)2 D3. This finding points to a novel molecular factor made by defective osteoblasts that could be involved in LPS-induced osteolysis.[unreadable] [unreadable] SLRP DEFICIENCIES AFFECT CRANIOFACIAL AND TOOTH FORMATION (50%)[unreadable] To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan, in tooth formation, we first investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium, and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.[unreadable] [unreadable] Biglycan (Bgn) and decorin (Dcn) are highly expressed in numerous tissues in the craniofacial complex. However, their expression and function in the cranial sutures is unknown. In order to study this, we first examined the expression of biglycan and decorin in the posterior frontal suture (PFS), which predictably fuses between 21 and 45 days post-natal, and in the non-fusing sagittal (S) suture from wild-type (Wt) mice. Our data showed that Bgn and Dcn were expressed in both cranial sutures. We then characterized the cranial suture phenotype in Bgn deficient, Dcn deficient, Bgn/Dcn double deficient, and Wt mice. At embryonic day 18.5, alizarin red/alcian blue staining showed that the Bgn/Dcn double deficient mice had hypomineralization of the frontal and parietal craniofacial bones. Histological analysis of adult mice (45-60 days post-natal) showed that the Bgn or Dcn deficient mice had no cranial suture abnormalities and immunohistochemistry staining showed increased production of Dcn in the PFS from Bgn deficient mice. To test for possible compensation of Dcn in the Bgn deficient sutures, we examined the Bgn/Dcn double deficient mice and found that they had impaired fusion of the PFS. Semi-quantitative RT-PCR analysis of RNA from 35 day-old mice revealed increased expression of Bmp-4 and Dlx-5 in the PFS compared to their non-fusing S suture in Wt tissues and decreased expression of Dlx-5 in both PFS and S in the Bgn/Dcn double deficient mice compared to the Wt mice. Failure of PFS fusion and hypomineralization of the calvaria in the Bgn/Dcn double deficient mice demonstrates that these extracellular matrix proteoglycans could have a role in controlling the formation and growth of the cranial vault.