Our major research interest is to study the regulatory mechanisms by which pluripotent mesenchymal stem cells differentiate into osteoblasts (bone-forming cells) or chondrocytes (cartilage-forming cells) and their roles in bone/cartilage regeneration and specific diseases. Currently, research in our laboratory is focused on the following projects:
Project 1: The role of bone sialoprotein (BSP) in osteoblast differentiation and bone regeneration
Bone sialoprotein (BSP) is one of the major non-collagenous extracellular matrix (ECM) glycophosphoproteins in bone and tooth. BSP is highly expressed in developing and postnatal regenerating bone, but its precise function is largely unclear. Our preliminary studies demonstrated that implantation of BSP with reconstituted type-I collagen, but not collagen alone, stimulated osteoblast differentiation and bone formation (osteogenesis) during the repair of cranial bone defects. In contrast, no bone formation was observed when BSP-collagen was implanted into the thoracic subcutaneous soft tissues (Figures 1 and 2).
Figure 1. Implantation of purified BSP (extracted from bovine bone, WB = Western blotting) with type-I collagen as a carrier into surgically created rat cranial bone defects and thoracic subcutaneous pouches.
Figure 2. BSP-collagen, but not collagen alone, stimulates new bone formation in the central regions of rat cranial bone defects. However, no bone formation was observed when BSP-collagen was implanted into the thoracic subcutaneous soft tissues. Note the new bone (nb) synthesized in the bone defect is distinguishable from the host bone (arrow) and from the periosteum-derived new bone (pnb).
The osteogenic bioactivity of BSP in stimulating bone regeneration is an unprecedented result; the mechanisms of tissue-specific BSP action remain unclear. The major goal of this project is to explore the molecular and cellular mechanisms of tissue-specific BSP action in osteogenesis and its application for enhancing bone regeneration. Successful completion of this project may advance our understanding of the mechanisms of BSP-specific biological activities, which will possibly lead to improved methods for the treatment of bone defects in humans. Moreover, understanding the cell type- and bony environment-dependent effects of BSP on osteogenesis may provide new insights into the long-standing question of why metastasis of BSP-expressing tumors is preferential to bone. This study was initially supported by the Orthopaedic Research & Education Foundation (OREF, PI: Jinxi Wang), and is currently supported by an NIH/NIDCR grant (1 R01 DE018713, PI: Jinxi Wang).
Project 2: Pathogenetic mechanisms and novel therapeutics for osteoarthritis
Osteoarthritis (OA) is the most common form of joint disease in middle-aged and older populations. No proven pharmacologic therapy is currently available to prevent the initiation or reverse the progression of OA, largely because the pathogenetic mechanisms of OA remain unclear. Recently, we discovered that mice lacking transcription factor Nfat1 exhibit normal skeletal development but display most of the characteristics of human OA (Figure 3).
Figure 3. Nfat1-/- articular chondrocytes do not display OA-like cellular dysfunction until the young adult stage. Photomicrographs were stained with Safranin-O and fast green. Scale bar = 200 µm for all photomicrographs. ( A) At the E16.5 embryonic stage, developing Nfat1-/- hip, knee, and shoulder joints display no morphological abnormalities compared to WT joints. Cartilage is stained in red with Safranin-O. ( B) At postnatal 1 day, skeletal tissues stained with Alcian blue and Alizarin red show no morphological abnormalities in Nfat1-/- mice. ( C) At 1 month of age (1m), both WT and Nfat1-/- hip joints display normal development. Cellular and extracellular matrix (ECM) morphology of WT and Nfat1-/- articular is presented in magnified square on right. (D) Articular cartilage harvested from the femoral head of a 4-month (4m) WT mouse shows normal articular surface and chondrocyte morphology with rich Safranin-O staining (red). In contrast, a 4-month Nfat1-/- femoral head shows articular surface fibrillation (arrow heads) with focal loss of Safranin-O staining and chondrocyte clustering (arrows), a typical feature of OA. ( E) A 9-month Nfat1-/- patellar-femoral joint demonstrates articular cartilage degradation and the formation of chondro-osteophytes (arrows). Dotted lines indicate the surfaces of the original articular cartilage/bone. ( F) An 18-month Nfat1-/- hip shows complete loss of articular cartilage (arrowhead) with exposure of thickened subchondral bone (♦). acet, acetabulum; fh, femoral head.
To explore the biological mechanisms of Nfat1 deficiency-induced OA, we have conducted the following preliminary studies.
Age-dependent gene and protein expression of Nfat1
To explore the mechanisms by which OA-like dysfunction of articular chondrocytes initially appears in the adult stage but not in the developmental stage of Nfat1-/- mice, we examined the expression pattern of Nfat1 from embryonic age E16.5 to 6 months by qPCR and immunohistochemistry (IHC). em>Nfat1 mRNA expression in articular chondrocytes was lowest at E16.5, gradually elevated postnatally, and then maintained at a high level at the adult stage (2-6 months) that we examined (Figure 4A). Immunohistochemical analyses demonstrated that intracellular localization of Nfat1 protein was essentially undetectable in articular chondrocytes at E16.5 but highly expressed at the adult stage (Figure 4B).
Figure 4.Age-dependent expression of Nfat1 in articular chondrocytes. ( A) Temporal changes of Nfat1 mRNA in articular chondrocytes of WT mice determined by qPCR. n = 3 pooled RNA samples; each prepared from the articular cartilage of six femoral heads. * p < 0.05; ** p < 0.01; *** p < 0.001. ( B) IHC using a specific antibody against mouse Nfat1 (Santa Cruz) demonstrates that intracellular or intranuclear Nfat1 protein expression in WT articular chondrocytes was undetectable at E16.5 (the box covers the developing joint space of the hip), but highly expressed (brown) at 6 months (6m). Scale bar = 200 µm.
Age-dependent histone methylation in the promoter region of the Nfat1 gene. We next investigated potential regulatory mechanisms for age-dependent Nfat1 expression. DNA methylation and histone modification are the two best-studied epigenetic regulatory mechanisms of gene transcription and cellular function in eukaryotic cells. DNA methylation patterns are usually stable, which confer long-term epigenetic modifications. In contrast, histone modifications are mostly short-term changes and therefore are reversible. Since the expression pattern of Nfat1 appeared to be reversible changes, we examined age-dependent histone modifications in the promoter region of the Nfat1 gene by ChIP assays.
ChIP assays were performed using chromatin prepared from articular chondrocytes isolated from femoral head articular cartilage of E16.5, 2-month, and 6-month WT mice and antibodies against H3K4me2 (a histone code associated with transcriptional activation), as well as H3K9me2 and H3K27me3 (histone codes associated with transcriptional repression). The binding level of co-immunoprecipitated DNA associated with histone methylation in each assay was quantified by qPCR analyses using three different primers (P1-3) designed from the sequences around the transcription start site (TSS) of the Nfat1 gene. A primer pair designed from the untranscribed genomic region located on mouse chromosome 17 (Untr17) was used as a negative control (Figure 5A). qPCR analyses of the ChIP samples using P1-3 revealed an age-related increase in H3K4me2 (Figure 5B) and an age-related decrease in H3K9me2 (Figure 5C), but no age-related change in H3K27me3 (data not shown). In contrast, qPCR using Untr17 control primer pair showed no detectable changes of H3K4me2 or H3K9me2 in this genomic DNA region (Figure 5B-C, P-ctl), confirming the specificity of the amplified PCR products for Nfat1-specificDNA sequences that areco-immunoprecipitated with H3K4me2 or H3K9me2.
Nfat1-specific Pol-II binding levels were correlated with H3K4me2 levels at different ages, while Gapdh-specific histone methylation levels showed no significant changes with age (data not shown), which further confirmed the specificity of the age-related histone methylation at the Nfat1 promoter. These results suggest that age-dependent expression of Nfat1 is associated with dynamic changes in specific histone methylation.
Figure 5. Age-dependent expression of Nfat1 is associated with dynamic changes in specific histone methylation.
Knockdown of Lsd1 in E16.5 articular chondrocytes results in an upregulation of Nfat1 expression concomitant with increased H3K4me2 at the Nfat1 promoter. To determine whether age-dependent Nfat1 expression in articular chondrocytes is regulated by epigenetic histone modifications, we performed RNAi-mediated loss-of-function experiments to test if knockdown of a key enzyme for H3K4 demethylation in chondrocytes affects Nfat1 expression. Previous studies identified Lsd1 as a key histone demethylase for H3K4me2 (Shi Y, et al. Cell 2004; 119 :941-53) and our study revealed that Nfat1 expression in E16.5 articular chondrocytes was very low, both in in vivo and in primary cultures. Thus, we tested whether knockdown of Lsd1 in E16.5 articular chondrocytes can up-regulate Nfat1 expression through increased H3K4me2 in the Nfat1 promoter region. The results demonstrated that Lsd1 RNAi efficiently reduced Lsd1 expression in cultured E16.5 primary articular chondrocytes, as judged by qPCR and Western blotting. Concomitant with the decrease in Lsd1 expression, we observed increased H3K4me2 levels around the TSS of the Nfat1 promoter by ChIP assays and an increase (derepression) in Nfat1 expression by qPCR in Lsd1 RNAi-treated E16.5 articular chondrocytes . The results suggest that the transcription of Nfat1 gene in E16.5 articular chondrocytes is negatively regulated by Lsd1 through demethylation of H3K4me2 at the Nfat1 promoter (Rodova M. et al. J Bone Miner Res 2011; 26:1974-86).
Knockdown of Jhdm2a in 6-month articular chondrocytes causes decreased Nfat1 expression concomitant with increased H3K9me2 at the Nfat1 promoter. We next tested if knockdown of a key enzyme for H3K9 demethylation affects Nfat1 expression in articular chondrocytes. Previous studies identified Jhdm2a as a specific histone demethylase for H3K9me2 (Yamane K, et al. Cell 2006;125 :483-95) and this study demonstrated that Nfat1 was highly expressed in 6-month articular chondrocytes, both in in vivo and in primary cultures. Thus, we examined whether knockdown of Jhdm2a in 6-month articular chondrocytes can down-regulate Nfat1 expression through increased H3K9me2. The results confirmed that Jhdm2a RNAi efficiently reduced Jhdm2a expression in cultured 6-month primary articular chondrocytes, as judged by qPCR (Figure 6A, left panel) and Western blotting (Figure 6A, right panel). Concomitant with the decrease in Jhdm2a expression, we observed increased H3K9me2 levels around the TSS of the Nfat1 promoter by ChIP assays (Figure 6B) and a decrease in Nfat1 expression by qPCR analyses (Figure 6C) in Jhdm2a RNAi-treated 6-month articular chondrocytes . The data suggest that the transcription of Nfat1 gene in 6-month articular chondrocytes is positively regulated by Jhdm2a through demethylation of H3K9me2 at the Nfat1 promoter (Figure 6D).
Taken together, these results demonstrate that age-dependent Nfat1 expression in articular chondrocytes is regulated by dynamic histone methylation, one of the epigenetic mechanisms in eukaryotic cells.
Figure 6. The transcription of Nfat1 gene in 6-month articular chondrocytes is positively regulated by Jhdm2a through demethylation of H3K9me2 at the Nfat1 promoter.
Our future studies will further explore the molecular and cellular mechanisms underlying the pathogenesis of NFAT1 deficiency-induced OA in adult mice and humans. This project is currently supported by an NIH R01 grant (1 R01AR059088, PI: Jinxi Wang).
Project 3: Regulatory mechanisms of chondrocyte function and articular cartilage regeneration
Currently, efforts to repair damaged articular cartilage face major obstacles due to limited intrinsic repair capacity of the tissue. Animal and human studies have demonstrated that a full thickness defect of articular cartilage penetrated through the subchondral bone to the bone marrow spaces can be repaired morphologically through the proliferation and differentiation of bone marrow stem cells into cartilage cells which synthesize a cartilage matrix, or by implanting chondrocyte-seeded biomaterials with or without growth factors using tissue engineering technology. However, the repaired articular cartilage tissue degenerates with reduced expression levels of cartilage markers after several months, and the joints with articular cartilage lesions eventually develop osteoarthritis.
The objective of this project is to investigate the regulatory mechanisms of adult chondrocyte function and articular cartilage homeostasis, thereby developing more effective therapeutic strategies for the healing of articular cartilage lesions.