Dayong Guo

Cilia-like structures and polycystin-1 in osteoblasts/ osteocytes and associated abnormalities in skeletogenesis and Runx2 expression

We examined the osteoblast/osteocyte expression and function of polycystin-1 (PC1), a transmembrane protein that is a component of the polycystin-2 (PC2)-ciliary mechano-sensor complex in renal epithelial cells. We found that MC3T3-E1 osteoblasts and MLO-Y4 osteocytes express transcripts for PC1, PC2, and the ciliary proteins Tg737 and Kif3a. Immunohistochemical analysis detected cilia-like structures in MC3T3-E1 osteoblastic and MLO-Y4 osteocyte-like cell lines as well as primary osteocytes and osteoblasts from calvaria. Pkd1m1Bei mice have inactivating missense mutations of Pkd1 gene that encode PC1. Pkd1m1Bei homozygous mutant mice demonstrated delayed endochondral and intramembranous bone formation, whereas heterozygous Pkd1m1Bei mutant mice had osteopenia caused by reduced osteoblastic function. Heterozygous and homozygous Pkd1m1Bei mutant mice displayed a gene dose-dependent decrease in the expression of Runx2 and osteoblast-related genes. In addition, overexpression of constitutively active PC1 C-terminal constructs in MC3T3-E1 osteoblasts resulted in an increase in Runx2 P1 promoter activity and endogenous Runx2 expression as well as an increase in osteoblast differentiation markers. Conversely, osteoblasts derived from Pkd1m1Bei homozygous mutant mice had significant reductions in endogenous Runx2 expression, osteoblastic markers, and differentiation capacity ex vivo. Co-expression of constitutively active PC1 C-terminal construct into Pkd1m1Bei homozygous osteoblasts was sufficient to normalize Runx2 P1 promoter activity. These findings are consistent with a possible functional role of cilia and PC1 in anabolic signaling in osteoblasts/osteocytes.

Transcriptional regulation of BMP-2 activated genes in osteoblasts using gene expression microarray analysis: role of Dlx2 and Dlx5 transcription factors.

This presentation will focus on using microarray data on a clonal osteoblast cell model to analyze the early BMP-2 responsive genes, as well as some of the later genes regulated by BMP2 during different phases of mineralization. We will focus on the early phases of gene expression that occur after BMP2 signaling from 30 min up to 1 day. The hypothesis is that understanding how these early genes are regulated during the initial multilayering and growth phase of osteoblasts will lead to models of how BMP activity stimulates cell growth, cell migration, multilayering, matrix deposition and remodeling phase that allows subsequent mineralization. The Dlx2 and Dlx5 homeobox genes have been shown to be critical for bone formation both in vitro and in vivo. Both Dlx 2 and Dlx5 are activated within 15-30 minutes after BMP2 addition to the mouse 2T3 osteoblast model and primary fetal rat calvarial osteoblasts. The Dlx2 and Dlx5 genes stay elevated in the presence of BMP2 for up to 5 days, a time when overt mineralization is just beginning. To understand the genomic network that Dlx5 and Dlx2 regulate at the transcription level, we have taken an approach where we use a specific transcription repressor protein, Engrailed, ligated to the Dlx5 homeodomain. The idea is that this Eng-Dlx5 protein will interact with Dlx5 and possibly Dlx2 and related Dlx- regulated genes in vivo and down-regulate their transcriptional initiation. Using a microarray approach with over 5,000 known genes we can identify the genes that are directly and indirectly regulated by Dlx5 and Dlx2. This will allow us to build an initial genomic network of Dlx- regulated genes at the transcriptional level. We will present our model and preliminary efforts at understanding the genomic network regulated by this important BMP2-regulated transcription factor class in osteoblast biology.

Gene expression signatures of a fibroblastoid preosteoblast and cuboidal osteoblast cell model compared to the MLO-Y4 osteocyte cell model

In the osteoblast 2T3 cell model, 326 genes significantly increase in expression as subconfluent fibroblastic 2T3 cells become confluent and cuboidal. This gene set includes BMP2/4, Dlx2/5, Runx2, Osterix and Lrp5, as well as TGFbeta regulated genes. Both activated or total nuclear Smad158 and Smad2 levels increase as they become confluent, and beta-catenin protein expression increases as 2T3 cells become confluent, reflecting a set of genes involved in early preosteoblast to osteoblast commitment, as observed in vitro and in vivo. Gene Set Enrichment Analysis (GSEA) demonstrated that this 326 dataset is very similar to several early osteoblast geneset signatures. The MLO-Y4 cell model is a well-known in vitro osteocyte model. The MLO-Y4 expression pattern was directly compared with the 2T3 osteoblast cell model. 181 genes that are highly expressed in MLO-Y4 osteocytes compared to osteoblasts were identified. Very few genes expressed in MLO-Y4 cells are found in osteocytes directly isolate from bone, suggesting that osteocyte specific gene programs most likely require the osteocytes to be embedded in the proper mineralized matrix. The MLO-Y4 dataset includes few established in vivo osteocyte markers, but does include several transcription factors such as Vitamin D receptor, Tcf7, and Irx5, whose expression was confirmed in osteocytes in vivo. Gene expression signatures in MLO-Y4 cells, as determined by functional clustering and interaction maps, suggest active prostaglandin-PKA pathways, genes involved in dendrite formation, acute/defense response pathways, TGFbeta signaling, and interferon/chemokine pathways. GSEA demonstrated that MLO-Y4 expression pattern is similar to macrophages, mesenchymal fibroblasts, and early osteoblasts.

New roles and mechanism of action of BMP4 in postnatal tooth cytodifferentiation

During the phase of overt tooth cytodifferentiation that occurs after birth in the mouse and using the 3.6Collagen1a-Cre and the BMP4 floxed and BMP4 knockout mice, the BMP4 gene was deleted in early collagen producing odontoblasts around postnatal day 1. BMP4 expression was reduced over 90% in alveolar osteoblasts and odontoblasts. There was decreased rate of predentin to dentin formation and decreased mature odontoblast differentiation reflected in reduced DMP1 expression and proper dentinal tubule formation, as well as reduced Collagen type I and Osteocalcin expression. We observed mutant dysmorphogenic odontoblasts that failed to properly elongate and differentiate. The consequence of this failed differentiation process leads to permanent loss of dentin thickness, apparent enlarged pulp chambers in the molars and reduced bone supporting the tooth structures in mice as old as 10-12 months. Deletion of the BMP4 gene in odontoblasts also indirectly disrupted the process of enamel formation that persisted throughout life. The mechanism for this altered differentiation program in the absence of the BMP4 gene in odontoblasts is from decreased BMP signaling, and decreased expression of three key transcription factors, Dlx3, Dlx5, and Osterix. BMP signaling, as well as Dlx3 and Amelogenin expression, is also indirectly reduced in the ameloblasts of the odontoblast BMP4 cKO mice. This supports a key paracrine or endocrine postnatal role of odontoblast derived BMP4 on the proper amelogenesis and formation of the enamel.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells

Summary We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKOob) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKOob mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKOob osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (&agr;-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in &agr;-SMA+ MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

Influences of reduced expression of maternal bone morphogenetic protein 2 on mouse embryonic development.

Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. In the course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3′ untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal mouse tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/–) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. The number of embryos exhibiting these abnormalities was increased when, due to different genotypes, expression levels of Bmp2 in maternal tissues were lower. These results suggest that the expression levels of Bmp2 in both embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds.

Dissection of promoter control modules that direct Bmp4 expression in the epithelium-derived components of hair follicles.

Bone morphogenetic protein 4 (Bmp4) plays a significant role in development. Using transgenic approaches, we studied the mechanisms controlling Bmp4 expression during primordial and mature tissue development, as well as in epithelium- and mesenchyme-derived components with hair follicles as a model. In this report, we demonstrated that the promoter region between the -0.26 and the -1.14 kb, highly conserved between human and mouse, controls Bmp4 expression in the epithelium-derived tissues but not in mesenchyme-derived tissues of hair follicles, suggesting that control modules for Bmp4 expression in epithelium-derived tissues and mesenchyme originated tissues are in separate regions. Using live embryos and mice, we demonstrated the temporal and spatial activities of these modules. We also demonstrated that control regions for Bmp4 expression in primordial and differentiated hair follicle tissues are separated. Therefore we present a model to explain a mechanism controlling expression of the Bmp4 gene in different tissue types, as well as different development stages as related to hair development.

Identification of osteocyte-selective proteins

Since little is known regarding osteocytes, cells embedded within the mineralized bone matrix, a proteomics approach was used to discover proteins more highly expressed in osteocytes than in osteoblasts to determine osteocyte‐specific function. Two proteomic profiles obtained by two different proteomic approaches using total cell lysates from the osteocyte cell line MLO‐Y4 and the osteoblast cell line MC3T3 revealed unique differences. Three protein clusters, one related to glycolysis (Phosphoglycerate kinase 1, fructose‐bisphosphate aldolase A, hypoxia up‐regulated 1 [ORP150], triosephosphate isomerase), one to protein folding (Mitochondrial Stress‐70 protein, ORP150, Endoplasmin), and one to actin cytoskeleton regulation (Macrophage‐capping protein [CapG], destrin, forms of lamin A and vimentin) were identified. Higher protein expression of ORP‐150, Cap G, and destrin in MLO‐Y4 cells compared with MC3T3 cells was validated by gene expression, Western blotting, and in vivo expression. These proteins were shown to be selective in osteocytes in vivo using immuno‐staining of mouse ulnae. Destrin was most highly expressed in embedding osteoid osteocytes, GapG in embedded osteocytes, and ORP150 in deeply embedded osteocytes. In summary, the proteomic approach has yielded important information regarding molecular mechanisms used by osteocytes for embedding in matrix, the formation of dendritic processes, and protection within a hypoxic environment.

Identification of Cis-DNA Regions Controlling Bmp4 Expression during Tooth Morphogenesis in vivo:

Epithelial-mesenchymal interactions are required for tooth formation. Bone morphogenetic protein 4 (Bmp4) is a crucial signaling molecule during this process. For better understanding of the role of the Bmp4 gene during tooth development, we studied the mechanisms that control its temporal and spatial expression during development. Using a transgenic approach, we determined that the domains which controlled Bmp4 expression in epithelium-derived ameloblasts were located in the region between 0.26 kb and 1.1 kb of the murine Bmp4 promoter. In contrast, the domains controlling Bmp4 expression in mesenchyme-derived odontoblasts and pulp cells existed in other regions of the Bmp4 gene. We have also demonstrated that the domains controlling Bmp4 expression in primordial tooth cells differ from those controlling Bmp4 expression in mature tooth tissues. The determination of unique domains by controlling the expression of the Bmp4 gene suggests that different transcriptional factors regulate the Bmp4 level at different stages during tooth morphogenesis.

Role of Bone Morphogenetic Proteins on Cochlear Hair Cell Formation: Analyses of Noggin and Bmp2 Mutant Mice

The mammalian organ of Corti of the inner ear is a highly sophisticated sensory end organ responsible for detecting sound. Noggin is a secreted glycoprotein, which antagonizes bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4). The lack of this antagonist causes increased rows of inner and outer hair cells in the organ of Corti. In mice, Bmp2 is expressed transiently in nascent cochlear hair cells. To investigate whether Noggin normally modulates the levels of Bmp2 for hair cell formation, we deleted Bmp2 in the cochlear hair cells using two cre strains, Foxg1cre/+ and Gfi1cre/+. Bmp2 conditional knockout cochleae generated using these two cre strains show normal hair cells. Furthermore, Gfi1cre/+;Bmp2lox/− mice are viable and have largely normal hearing. The combined results of Noggin and Bmp2 mutants suggest that Noggin is likely to regulate other Bmps in the cochlea such as Bmp4. Developmental Dynamics 239:505–513, 2010. Published 2010 Wiley‐Liss, Inc.