Function Of Skeletal Matrix Genes

Dental & Craniofacial Research

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Abstract

The goal of the Section is to explore the function of matrix proteins made in skeletal tissues. Our strategy is to focus on a single category of Extracellular Matrix (ECM) proteins known as small leucine rich proteoglycans (SLRPs) that are highly expressed in bones and teeth. [unreadable] [unreadable] BIGLYCAN DEFICIENCY INCREASES OSTEOCLAST DIFFERENTIATION AND ACTIVITY DUE TO DEFECTIVE OSTEOBLASTS AND CAUSES GENDER AND BONE SPECIFIC SKELETAL DEFECTS We have previously shown that biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. This year, we showed 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 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 Bgn 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 bgn deficient mice than wt 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 RANKL and 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 decrease in SLPI (secretory leukocyte protease inhibitor) in Bgn 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] In order to understand the role of Bgn on bone's geometric and mechanical properties, the normal and bgn-deficient mice were assayed by four-point bending tests as well as for their bone geometry and bone formation using histomorphometry. Interestingly, Bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure, suggesting a new functional role of Bgn in regulating bone mineralization in vivo.[unreadable] [unreadable] SLRP DEFICIENCIES AFFECT TOOTH FORMATION In order to determine the role that biglycan and other SLRPs might play in craniofacial tissues, the teeth of normal and biglycan or decorin KO mice were examined by histology and electron microscopy. Each deficiency resulted in specific disruptions of dentin and enamel formation. Biglycan and decorin deficiencies had the same overall effect on dentin formation, hypomineralized dentin, although this effect was more dramatic in the absence of decorin. In contrast, biglycan and decorin deficiencies had opposite effects on enamel formation. Enamel formation was dramatically increased in biglycan-deficient mice, whereas it was decreased in the absence of decorin. The increased enamel formation in the absence of biglycan correlated with an up-regulation of amelogenin synthesis, in contrast to decreased enamel formation in the absence of decorin. The decorin KO phenotype was possibly an indirect consequence of the disruption in dentin formation, dentin being so porous that it prevented initial enamel deposition. In conclusion, our results indicate that biglycan and decorin control late stages of tooth formation. The phenotypes of biglycan- and decorin-deficient mice indicate that during tooth development, biglycan represses amelogenin expression and enamel formation, whereas decorin promotes dentin. Other studies using fibromodulin knockout mice showed that, in contrast to biglycan and decorin KO mice, they had increased dentin with no effects on enamel formation. Taken together, a complex picture is emerging where individual SLRPS can have positive and negative effects on mineralized tooth tissue formation, depending on the SLRP and its in situ tissue milieu. [unreadable] [unreadable] To determine the functions of fibromodulin (Fmod), we first investigated its distribution in dental tissues by immunohistochemistry and then 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] Hydroxyapatite mineral is deposited in an organized fashion in the matrices of bones and teeth. This year we reviewed methods for measuring mineral properties and provide some illustrations from bones and teeth of animals in which the small leucine-rich proteoglycans (bgn and decorin) were ablated. Our data showed there were mineralization defects that were dependent on genotype and tissue origin. This observation points to the concept that the mechanisms regulating mineralization could be controlled by the unique SLRP ECM millieu found in bones and teeth.[unreadable] [unreadable] ACCELERATED TEMPORAL MANDIBULAR JOINT OSTEOARTHRITIS IN THE BIGLYCAN/FIBROMODULIN DOUBLE KNOCKOUT MICE The temporomandibular joint (TMJ) within the craniofacial complex is unique. In humans, the TMJ can become diseased resulting in severe and disabling pain. There are no cures for TMJ disease at this time. Animal models of TMJ disease are scarce, but some exist, and they are described in a review published this year. We present in greater detail one animal model that is deficient in two extracellular matrix (ECM) proteoglycans, biglycan (BGN) and fibromodulin (FMOD). Doubly deficient BGN/FMOD mice develop premature TMJ osteoarthritis (OA). In order to explore the mechanistic basis of TMJ-OA, tissues from the condyle of mutant mice were examined for their relative capacity to differentiate and undergo apoptosis. Our data show that there is a redistribution of the critical ECM protein, type II collagen, in mutant mice compared with controls. Mutant mice also have increased apoptosis of the chondrocytes embedded in the articular cartilage. We speculate that the overall imbalance in apoptosis may be the cellular basis for the abnormal production of extracellular matrix proteins. The abnormal production of the ECM could, in turn, lead to premature erosion and degradation of the articular surface resulting in TMJ-OA.