Qiping Zheng

Runx2 contributes to murine Col10a1 gene regulation through direct interaction with its cis-enhancer

We have recently shown that a 150‐bp Col10a1 distal promoter (−4296 to −4147 bp) is sufficient to direct hypertrophic chondrocyte‐specific reporter (LacZ) expression in vivo. More recently, through detailed sequence analysis we identified two putative tandem‐repeat Runx2 binding sites within the 3′‐end of this 150‐bp region (TGTGGG‐TGTGGC, −4187 to −4176 bp). Candidate electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation, and transfection studies demonstrate that these putative Runx2 sites bind Runx2 and mediate upregulated Col10a1/reporter activity in vitro. Transgenic studies using the 5′‐sequence without Runx2 sites were not able to drive the cell‐specific LacZ reporter activity, suggesting the in vivo requirement of the Runx2 sites located in the 3′‐end in mediating Col10a1/reporter expression. Indeed, mutating the Runx2 sites in the context of the 150‐bp promoter abolishes its capacity to drive hypertrophic chondrocyte‐specific reporter expression in transgenic mice. We have also generated multiple transgenic mouse lines using only the 3′‐sequence containing the Runx2 sites to drive the LacZ gene. Interestingly, no hypertrophic chondrocyte‐specific blue staining was observed in these transgenic mice. Together, our data support that Runx2 directly interacts with murine Col10a1 cis‐enhancer. This interaction is required but not sufficient for cell‐specific Col10a1 promoter activity in vivo. Additional cooperative/repressive elements within the 5′‐ or 3′‐sequences of this 150‐bp promoter are needed to work with Runx2 together to mediate cell‐specific Col10a1 expression. Further delineation of these elements/factors has the potential to identify novel therapeutic targets for multiple skeletal disorders, including osteoarthritis, that show abnormal Col10a1 expression and altered chondrocyte maturation.

TARGETING RUNX2 EXPRESSION IN HYPERTROPHIC CHONDROCYTES IMPAIRS ENDOCHONDRAL OSSIFICATION DURING EARLY SKELETAL DEVELOPMENT

Runx2 is a known master transcription factor for osteoblast differentiation, as well as an essential regulator for chondrocyte maturation. Recently, more and more data has shown that Runx2 regulates hypertrophic chondrocyte‐specific type X collagen gene (Col10a1) expression in different species. However, how Runx2 regulation of Col10a1 expression impacts chondrocyte maturation, an essential step of endochondral bone formation, remains unknown. We have recently generated transgenic mice in which Flag‐tagged Runx2 was driven by a cell‐specific Col10a1 control element. Significantly increased level of Runx2 and Col10a1 mRNA transcripts were detected in transgenic mouse limbs at both E17.5 (embryonic day 17.5) and P1 (post‐natal day1) stages, suggesting an in vivo correlation of Runx2 and Col10a1 expression. Surprisingly, skeletal staining suggested delayed ossification in both the axial and the appendicular skeleton of transgenic mice from E14.5 until P6. Histological analysis showed elongated hypertrophic zones in transgenic mice, with less von Kossa and TUNEL staining in long bone sections at both E17.5 and P1 stages, suggesting defective mineralization due to delayed chondrocyte maturation or apoptosis. Indeed, we detected increased level of anti‐apoptotic genes B‐cell leukemia/lymphoma 2, Osteopontin, and Sox9 in transgenic mice by real‐time RT‐PCR. Moreover, immunohistochemistry and Western blotting analysis also suggested increased Sox9 expression in hypertrophic chondrocytes of transgenic mice. Together, our data suggest that targeting Runx2 in hypertrophic chondrocytes upregulates expression of Col10a1 and other marker genes (such as Sox9). This will change the local matrix environment, delay chondrocyte maturation, reduce apoptosis and matrix mineralization, and eventually, lead to impaired endochondral ossification. J. Cell. Physiol. 227: 3446–3456, 2012.

MALAT1 promotes osteosarcoma development by targeting TGFA via MIR376A

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) that contributes to the initiation and development of many solid tumors, including osteosarcoma (OS). Here, we showed that MALAT1 was increased in human OS cell lines and tissues and promoted OS cell growth, while MALAT1 knockdown suppressed OS cell growth. We also detected downregulation of MIR376A, a suppressor of OS growth, and upregulation of TGFA, a promoter of OS growth, in OS tissues. TGFA expression was positively correlated with MALAT1 expression, and both were negatively correlated with MIR376A expression. There was a direct interaction between MIR376A and MALAT1 via a putative MIR376A binding site within the MALAT1 3′-untranslated region (3′-UTR). There was also a direct interaction between MIR376A and the TGFA 3′-UTR. Thus, MALAT1 may promote OS cell growth through inhibition of MIR376A, leading to increased expression of TGFA. Our results suggest a MALAT1/MIR376A/TGFA axis mediates OS cell proliferation and tumor progression.

Distinct function of P63 isoforms during embryonic skeletal development

P63 belongs to the P53 family of transcription factors. There are multiple P63 isoforms that play important functions both in cancer and development. The obvious limb defect in p63 null mice and in human skeletal syndromes with P63 mutations suggest its essential role in long bone development. However, how the different P63 isoforms function during long bone development is largely unknown. We have previously shown that TAP63α, the longest P63 isoform, plays a positive role in embryonic skeletal development, since targeting TAP63α expression in hypertrophic chondrocytes accelerates endochondral ossification at both E17.5 and P1 stages. Here, we report transgenic studies of ΔNP63α, another P63 isoform which lacks the N-terminal transactivation domain compared to TAP63α, using the same hypertrophic chondrocyte-specific Col10a1 control element. No skeletal abnormalities were detected in these Col10a1-ΔNP63α transgenic mice at both E17.5 and P1 stages, suggesting less importance of ΔNP63α during late embryonic skeletal development. To further investigate the function of P63 isoforms during early skeletal development, we have generated ΔNP63α and TAP63α transgenic mice using a chondrocyte-specific Col2a1 control element. Surprisingly, while no skeletal defect was shown in the Col2a1-ΔNP63α transgenic mice, reduced ossification was observed in the digit and tail bones of Col2a1-TAP63α transgenic mice at both E17.5 and P1 stages compared to their wild-type littermates. Expression profiling and immunohistochemical analysis detected upregulated expression of Sox9, a major negative regulator of endochondral ossification, in Col2a1-TAP63α transgenic mice. Taken together, our results suggest a distinct function of P63 isoforms, herein, ΔNP63α and TAP63α, during endochondral ossification.

RUNX2 and Osteosarcoma

Osteosarcoma (OS) is the most common pediatric bone cancer in children and young adults. Previous studies have suggested the importance of osteoblast activity in OS tumorigenesis and metastasis, as OS is characterized by abnormal bone formation, while osteoblast is the predominant cell type both in OS and in metastatic tumor tissues. RUNX2 is a known essential transcription factor for osteoblast differentiation. RUNX2 has also been linked to many human cancers, including bone cancers and cancer metastasis in bone. However, the view of RUNX2 during OS tumorigenesis has not been unanimous. In this manuscript, we reviewed the osteoblastic origin in OS etiology. The oncogenic property of RUNX2 in human OS studies was briefly summarized. RUNX2 may be involved in OS pathogenesis by regulating cell cycle controlling of (pre)-osteoblasts, which subsequently convert to OS cells. The roles and mechanisms of RUNX2 during OS metastasis and bone metastasis in target cancers (herein prostate and breast cancers), were as described. The potential involvement of Runx2 in multiple mouse OS models that use human OS cell lines (Xenografts), tumor suppressor genes p53 and Rb1 were also discussed. Finally, we updated some microRNAs studies and their relation with RUNX2 in OS pathogenesis. This review provides a comprehensive understanding of RUNX2s function during OS pathogenesis and will help with the research designing and strategy in controlling OS.

Putative function of TAP63α during endochondral bone formation.

P63, a member of the P53 tumor suppressor family, is known to play important functions in cancer and development. Interestingly, previous studies have shown that p63 null mice are absent or have truncated limbs, while mutations in human P63 cause several skeletal syndromes that also show limb and digit abnormalities, suggesting its essential role in long bone development. Indeed, we detected increased level of p63 transcript in hypertrophic MCT cells (an established cell model of chondrocyte maturation) than in proliferative MCT cells. To investigate the in vivo role of P63 upon endochondral bone formation, we have established transgenic mouse lines in which HA- and Flag-tagged TAP63α (the longest P63 isoform) is driven by the hypertrophic chondrocyte-specific Col10a1 regulatory elements. Skeletal staining of Col10a1-TAP63α transgenic mice at either embryonic day 17.5 (E17.5) or postnatal day 1 (P1) observed accelerated ossification in long bone, digit and tail bones compared to their wild-type littermates, suggesting a putative function of P63 during skeletal development. We also detected decreased level of Sox9 and Bcl-2 transcripts, while Alp and Ank are slightly upregulated in Col10a1-TAP63α transgenic mouse limbs. Further immunohistochemical analysis confirmed the decreased Sox9 expression in the proliferative and hypertrophic zone of these mice. Von Kossa staining suggests increased mineralization in hypertrophic zone of transgenic mice compared to littermate controls. Together, our results suggest a role of TAP63α upon skeletal development. TAP63a may promote endochondral ossification through interaction with genes relevant to matrix mineralization and chondrocyte maturation or apoptosis.

TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

BACKGROUND/AIMS TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. METHODS We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC(KM)) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by (3)H-TdR incorporation. RESULTS TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. CONCLUSIONS TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.

Col10a1 gene expression and chondrocyte hypertrophy during skeletal development and disease

The type X collagen gene, COL10A1, is specifically expressed by hypertrophic chondrocytes during endochondral ossification. Endochondral ossification is a well-coordinated process that involves a cartilage intermediate and leads to formation of most of the skeleton in vertebrates during skeletogenesis. Chondrocyte hypertrophy is a critical stage of endochondral ossification linking both bone and cartilage development. Given its specific association with chondrocyte hypertrophy, type X collagen plays essential roles in endochondral ossification. It was previously shown that transgenic mice with mutant type X collagen develop variable skeleton-hematopoietic abnormalities indicating defective endochondral ossification, while mutations and abnormal expression of human COL10A1 cause abnormal chondrocyte hypertrophy that has been seen in many skeletal disorders, including skeletal chondrodysplasia and osteoarthritis. In this review, we summarized the skeletal chondrodysplasia with COL10A1 gene mutation that shows growth plate defect. We also reviewed recent studies that correlate the type X collagen gene expression and chondrocyte hypertrophy with osteoarthritis. Due to its significant clinical relevance, the type X collagen gene regulation has been extensively studied over the past two decades. Here, we focus on recent progress characterizing the cis-enhancer elements and their binding factors that together confer hypertrophic chondrocyte-specific murine type X collagen gene (Col10a1) expression. Based on literature review and our own studies, we surmise that there are multiple factors that contribute to hypertrophic chondrocyte-specific Col10a1 expression. These factors include both transactivators (such as Runx2, MEF2C etc.) and repressors (such as AP1, NFATc1, Sox9 etc.), while other co-factors or epigenetic control of Col10a1 expression may not be excluded.

In vitro functional characterization of prostaglandin-endoperoxide synthase 2 during chondrocyte hypertrophic differentiation.

Cyclooxygenase 2 (Cox-2) has been implicated an essential role during bone repair, but the mechanisms remain elusive. Bone repair healing is known to include processes similar to endochondral ossification. In this study, we investigated the in vitro effect of Cox-2 on Col10a1 expression and chondrocyte hypertrophy, two critical components of endochondral ossification. Using quantitative RT-PCR, we detected increased mRNA levels of Cox-2 and Col10a1 in hypertrophic MCT cells, while cells treated with Cox-2 inhibitor, NS398, showed decreased mRNA and protein levels of Cox-2 and Col10a1. Increased Cox-2 also correlated with significantly upregulated Col10a1 in hypertrophic ATDC5 cells, whereas inhibition of Cox-2 significantly decreased Col10a1 expression. We further generated a Cox-2-expressing ATDC5 stable cell line. Compared with the controls, Cox-2 over-expression significantly increased Col10a1 as early as day 7 of continuous culturing, but not at days 14 and 21. Enhanced Alp staining was also observed in day 7 stable cell line. Correspondingly, we detected significantly increased levels of Runx2, Alp, Bcl-2, Bax, Col1a1, Osterix, and Bsp in day 7 stable line. Most of these genes have been associated with chondrocyte maturation and apoptosis. Together, our results support that Cox-2 promotes Col10a1 expression and chondrocyte hypertrophy in vitro, possibly through upregulation of Runx2 and other relevant transcription factors.

Abstract 4972: Putative mechanism of TAP63α regulation of chondrocyte maturation during long bone development

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC p63 is the mouse homologue of the well-known tumor suppressor P53. While it remains controversial as to whether P63 functions as a tumor suppressor gene or an oncogene, mouse genetic studies and mutation analysis of human SHFM syndrome (Split Hand/Foot Malformation) have clearly demonstrated that P63 plays an important role in skin and limb development. The truncated limbs observed in p63 null mice suggest that P63 may contribute to long bone development by playing a role during endochondral bone formation. However, how P63 regulates target gene expression and is linked to endochondral ossification remains largely unknown. Here we present some preliminary data suggesting that P63 may regulate type X collagen gene (Col10a1) expression, thereby impacting the process of chondrocyte maturation. The type X collagen gene (Col10a1) is specifically expressed in hypertrophic chondrocytes, a critical cell stage linking both skeletal development and multiple skeletal diseases. We have previously shown that a 150-bp Col10a1 cis-enhancer is sufficient to mediate its cell-specific expression in vivo. Our further in vitro studies suggest that Runx2, a master transcription factor for osteoblast differentiation as well as for chondrocyte maturation, is one of the major factors that regulate Col10a1 expression via interaction with its tandem repeat Runx2 binding sites. Interestingly, by yeast one-hybrid approach using this cis-enhancer as bait, we identified p53 related proteins as candidate factors that may contribute to the regulation of Col10a1 expression. We have performed Real-time RT-PCR to examine p63 and Runx2 expression in MCT cells, a cell model that shows significant upregulation of Col10a1 upon growth arrest. Both p63 and Runx2 showed around 2-fold upregulation in hypertrophic MCT cells. These data suggest that P63 may collaborate with Runx2 and together regulate Col10a1 expression during chondrocyte maturation. To further explore the putative function of P63 during skeletal development, we have successfully established transgenic mouse lines in which HA and Flag tagged human TAP63α cDNA (the longest P63 isotype) is driven by previously described hypertrophic chondrocyte-specific Col10a1 control element (Col10a1-TAP63α). By phenotypic analysis of these transgenic mice during different developmental stages, we will be able to define the consequence and the potential mechanism of P63 on skeletal development and skeletal disorders. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4972.