Role of dentin matrix protein 1 in cartilage redifferentiation and osteoarthritis

Objective The aim of this study was to test the possible involvement, relevance and significance of dentin matrix protein 1 (DMP1) in chondrocyte redifferentiation and OA. Methods To examine the function of DMP1 in vitro, bone marrow stromal cells (BMSCs) and articular chondrocytes (ACs) were isolated and differentiated in micromasses in the presence or absence of DMP1 small interfering RNA and analysed for chondrogenic phenotype. The association of DMP1 expression with OA progression was analysed time dependently in the OA menisectomy rat model and in grade-specific OA human samples. Results It was found that DMP1 was strongly related to chondrogenesis, which was evidenced by the strong expression of DMP1 in the 14.5-day mouse embryonic cartilage development stage and in femoral heads of post-natal days 0 and 4. In vitro chondrogenesis in BMSCs and ACs was accompanied by a gradual increase in DMP1 expression at both the gene and protein levels. In addition, knockdown of DMP1 expression led to decreased chondrocyte marker genes, such as COL2A1, ACAN and SOX9, and an increase in the expression of COL10A and MMP13 in ACs. Moreover, treatment with IL-1β, a well-known catabolic culprit of proteoglycan matrix loss, significantly reduced the expression of DMP1. Furthermore, we also observed the suppression of DMP1 protein in a grade-specific manner in knee joint samples from patients with OA. In the menisectomy-induced OA model, an increase in the Mankin score was accompanied by the gradual loss of DMP1 expression. Conclusion Observations from this study suggest that DMP1 may play an important role in maintaining the chondrogenic phenotype and its possible involvement in altered cartilage matrix remodelling and degradation in disease conditions like OA.

Inflammation-driven bone formation in a mouse model of ankylosing spondylitis: Sequential not parallel processes

Background Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. Methods The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. Results Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease. Conclusions The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-beta 3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers

DMRT1 has been suggested to play different roles in sex determination and gonad differentiation, because different expression patterns have been reported among different vertebrates. The groupers, since their gonads first develop as ovary and then reverse into testis, have been thought as good models to study sex differentiation and determination. In this study, we cloned the full-length cDNAs of DMRT] gene from orange-spotted grouper (Epinephelus coioides), and prepared corresponding anti-EcDMRT1] antiserum to study the relationship of DMRT] to sex reversal. One important finding is that the grouper DMRT] is not only differentially expressed in different stage gonads, but also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, we confirmed that EcSox3 is expressed not only in oogonia and different stage oocytes, but also in Sertoli cells and spermatogonia, and EcSox9 is expressed only in Sertoli cells. The data suggested that grouper DMRT1 might be a more specific sex differentiation gene for spermatogenesis, and play its role at the specific stages from spermatogonia to spermatocytes. In addition, no introns were found in the grouper DMRT1, and no duplicated DMRT1, genes were detected. The finding implicates that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad. (c) 2006 Published by Elsevier Ireland Ltd.

非洲爪蟾Nkx6.3在原肠运动和神经嵴诱导调控中的功能及爪蟾神经管腹侧图式形成的研究

神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发现，在爪蟾胚胎中Nkx6.3的过表达或抑制表达都导致神经嵴标记基因表达降低。进一步研究发现，32细胞期在不同部位注射Nkx6.3 mRNA可以异位诱导或抑制Slug的表达。动物帽实验显示，Nkx6.3单独过表达可以诱导神经嵴发生，而迄今为止转录因子中只有Snail1具有这一单独诱导能力。在爪蟾胚胎及动物帽中，过表达Nkx6.3都可以诱导Fgf8、Wnt8而抑制BMP4的转录，而且Nkx6.3对这些分泌因子的调控方式是不同的。4细胞期过表达Nkx6.3的胚胎，在促进Fgf8和Wnt8而抑制BMP4的同时，却抑制神经板边界特异基因Msx1、Pax3和神经嵴特异基因Slug的表达，说明Nkx6.3对神经嵴的诱导调控在神经板边界基因层次还存在抑制作用。32细胞过表达Nkx6.3会细胞自主性抑制以及细胞非自主诱导Msx1、Pax3、Slug的表达。Nkx6.3异位诱导Dlx5却抑制Dlx3的表达，说明Dlx5可能是Nkx6.3负调控的直接靶基因。由此，我们提出Nkx6.3的神经嵴诱导调控分为两个层次：分泌信号分子水平的正调控和神经板边界决定水平的负调控。在脊椎动物的神经发生过程中，神经管背腹不同层次形成不同的神经元。这些神经元细胞的命运由背腹起源的多种形态发生素决定。形态发生素通过浓度梯度确定了一组转录因子在神经管背腹不同层次的特异表达，这些基因的组合调控决定了神经前体细胞的命运。然而，这些转录因子是如何解读形态发生素梯度信号的还不是很清楚。第四章，我们通过对神经管腹侧特异表达的转录因子的调控区进行预测，确定了可能调控这些基因表达的保守区段。此外，我们改进了爪蟾转基因操作，并用这一技术确证了Nkx6.2的调控区域。Dbx1、Nkx2.2及Pax6的转录调控区已在小鼠或爪蟾中报道过。由此我们得到了两对在神经管背腹图式中相互作用的转录因子的调控区域：Nkx6.2和Dbx1、Nkx2.2和Pax6。通过对Nkx6.2和Dbx1的调控保守区的转录因子结合位点的预测，我们发现这四个基因以及Wnt信号之间存在大量的相互调控。然而在这两个基因的调控区，我们没有发现Gli的调控位点，暗示这两个基因可能不受Shh的直接调控。我们还克隆了Dbx家族的两个基因，并检测了它们的时空特异性表达，发现Dbx2是母源性表达的，而Dbx1是合子型基因。这两个基因的表达图式相似，都在神经板中线两侧成线状表达，尾芽期在神经管中部表达。过表达Dbx2抑制神经元的初级分化，说明它可能与Dbx1一样具有维持神经板细胞未分化状态的功能。Dbx2的过表达还抑制Nkx6.2及Dbx1的表达，说明它们可能一起参与了神经管腹侧图式的调控。

非洲爪蛙分泌型叶酸结合蛋白及NKX6.3在早期胚胎神经发育中的功能研究

叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此我们认为sFBP结合叶酸后可能通过细胞膜上的受体传递信号，并且Islet-1可能在sFBP的下游发挥作用。 神经嵴是脊椎动物特有的一群多潜能干细胞，产生于表皮和神经板的边界，在原肠运动之后这群细胞通过表皮间充值转换从神经管背侧迁移到不同的区域，分化成不同的细胞类型，包括外周神经系统，色素细胞，软骨等。神经嵴的发生是一个多步骤多基因参与的精细调控过程。目前理论认为最初由一些分泌性信号分子又叫形态生成素比如BMP,Wnt，FGF，Notch等通过不同浓度梯度的相互作用调节一组在表皮和神经板边界的转录因子（Msx、Pax3/7、Zic1、Dlx3/5等）的表达，即边界决定。这些边界决定因子进一步在预定形成神经嵴的区域激活神经嵴特化基因比如Slug/Snail、FoxD3、Twist、Sox9/10的表达完成神经嵴的特化（Specification）。 Nkx6.3是Nkx6家族的一个转录因子，RT-PCR显示其呈现母源性表达。特异抗体显示Nkx6.3蛋白第9期在整个胚胎都表达，大部分蛋白集中在细胞核，有少部分蛋白定位于细胞膜上；神经板时期主要定位于神经嵴区域的细胞膜上。过表达Nkx6.3会影响细胞粘连分子的表达，由此干扰正常的胚胎原肠运动和Activin诱导的动物帽聚集延伸运动。显微注射Nkx6.3特异morpholino阻断其蛋白表达会抑制神经嵴的marker基因Wnt8，Fgf8，Pax3，Msx1，Zic1，FoxD3，Slug的转录，阻碍神经嵴的发育。在动物帽中单独注射Nkx6.3可以在mRNA水平上诱导Wnt8、Fgf8另一方面抑制BMP4的表达进而诱导神经嵴基因Pax3，Zic1，Slug的表达。报告基因实验也显示Nkx6.3能够激活Wnt信号而在动物帽中抑制BMP信号。Nkx6.3蛋白功能域分析发现其EH1结构域（domain）参与对Wnt8信号的激活，而EH1结构域和HD结构域之间的连接区域（linker domain）参与对FGF的激活和对BMP的抑制。进一步在动物帽和胚胎中分析发现Nkx6.3对Wnt8的激活依赖于FGF家族受体信号但是不依赖于Fgf8。有趣的是4细胞时期过表达Nkx6.3促进Fgf8和Wnt8 mRNA表达，但是抑制边界决定基因Msx1、Pax3和神经嵴特化基因Slug的转录。在32细胞时期显微注射Nkx6.3可以在内源神经嵴发生区域抑制Slug的表达，而异位却诱导Slug的mRNA。我们发现与动物帽中对BMP的调节不同，在胚胎中，过表达Nkx6.3会强烈的激活Smad1蛋白在细胞核中的表达即BMP信号被激活，高的BMP信号会抑制神经嵴的发生。另外我们发现过表达Nkx6.3在胚胎中抑制Dlx5而在动物帽中却不影响Dlx5的表达水平，Morpholino阻断Dlx5会抑制Msx1、Pax3和Slug的表达。BMP信号和Dlx5在动物帽和在整体胚胎中对Nkx6.3的不同响应可以一定程度上解释过表达Nkx6.3在2个系统中对神经嵴基因Slug相反的影响结果。

Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Evolution, gene regulation and functional analysis of BMP2 in fish

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.

Molecular characterization of Gla-rich protein (GRP) gene expression and function

Gla-rich protein (GRP) is a vitamin K-dependent protein related to bone and cartilage recently described. This protein is characterized by a large number of Gla (γ-carboxyglutamic acid) residues being the protein with the highest Gla content of any known protein. It was found in a widely variety of tissues but highest levels was found in skeletal and cartilaginous tissues. This small secreted protein was also expressed and accumulated in soft tissues and it was clearly associated with calcification pathologies in the same tissues. Although the biological importance of GRP remains to be elucidated, it was suggested a physiological role in cartilage development and calcification process during vertebrate skeleton formation. Using zebrafish, an accepted model to study skeletal development, we have described two grp paralog genes, grp1 and grp2, which exhibited distinct patterns of expression, suggesting different regulatory pathways for each gene. Gene synteny analysis showed that grp2 gene is more closely related to tetrapod grp, although grp1 gene was proposed to be the vertebrate ortholog by sequence comparison. In addition, we identified a functional promoter of grp2 gene and using a functional approach we confirmed the involvement of transcription factors from Sox family (Sox9b and Sox10) in the regulation of grp2 expression. In an effort to provide more information about the function of grp isoforms, we generated two zebrafish transgenic lines capable to overexpress conditionally grp genes and possible roles in the skeleton development were studied. To better understand GRP function a mammalian system was used and the analysis of knockout mice showed that GRP is involved in chondrocyte maturation and the absence of GRP is associated to proteoglycans loss in calcified articular cartilage. In addition, we detected differences in chondrogenesis markers in articular chondrocyte primary culture. Overall, our data suggest a main role for GRP on chondrocyte differentiation.

Effects of vitamin K deficiency and its mechanisms in vertebrate's early development: zebrafish as a model

Disertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Identification of the molecular sex-differentiation period in the siberian sturgeon

Sexual development prior to gonadal sex differentiation is regulated by various molecular mechanisms. In fish, a molecular sex-differentiation period has been identified in species for which sex can be ascertained prior to gonadal sex differentiation. The present study was designed to identify such a period in a species for which no genetic sex markers or monosex populations are available. Siberian sturgeons undergo a slow sex-differentiation process over several months, so gonad morphology and gene expression was tracked in fish from ages 3-27 months to identify the sex-differentiation period. The genes amh, sox9, and dmrt1 were selected as male gonad markers; cyp19a1a and foxl2a as female gonad markers; and cyp17a1 and ar as markers of steroid synthesis and steroid receptivity. Sex differentiation occurred at 8 months, and was preceded by a molecular sex-differentiation period at 3-4 months, at which time all of the genes except ar showed clear expression peaks. amh and sox9 expression seemed to be involved in male sexual development whereas dmrt1, a gene involved in testis development in metazoans, unexpectedly showed a pattern similar to those of the genes known to be involved in female gonadal sex differentiation (cyp19a1 and foxl2a). In conclusion, the timing of and gene candidates involved with molecular sex differentiation in the Siberian sturgeon were identified. Mol. Reprod. Dev. 2015. © 2015 Wiley Periodicals, Inc.

Conditional and transgenic mouse models as tools to study the role of TGFbeta, BMP signaling in skeletal development

Die TGFbeta/BMP Signaltransduktionskaskade ist wichtig für viele Entwicklungsprozesse fast aller embryonaler sowie extraembryonaler Gewebe und sie ist ebenso essentiell bei der Aufrechterhaltung der Homöostase im adulten Organismus. In vielen Mausmodellen und Zellkulturversuchen wurde gezeigt, dass Liganden dieses Signalweges in verschiedene Stadien der Knorpel- und Knochenentwicklung involviert sind. BMPs sind beispielsweise maßgeblich an der frühen Kondensation und Bildung des Knorpels und später an Proliferation und Hypertrophie der Chondrozyten beteiligt. BMPs können ektopisch Knochenbildung auslösen und das Expressionsmuster der Liganden und spezifischen Rezeptoren in der Wachstumsfuge lässt auf eine wichtige Rolle der BMPs in der Wachstumsfuge schließen. Der gezielte knock out der BMP-Rezeptoren Bmpr1a und Bmpr1b in proliferierenden Chondrozyten führt zur Ausbildung einer generellen Chondrodysplasie. Smad1, Smad5 und Smad8 sind die Mediatoren der BMP-Signalkaskade. Im Rahmen der vorliegenden Arbeit sollte die Rolle und Funktion der Smad1- und Smad5-Proteine in der Wachstumsfuge untersucht werden. Hierzu wurden konditionale Smad1-knock out-Mäuse mit einer transgenen Mauslinie gekreuzt, die die Cre-Rekombinase spezifisch in proliferierenden Chondrozyten exprimiert. Diese Mäuse wurden mit und ohne heterozygotem Smad5-Hintergrund charakterisiert. Bei einem knock out von Smad1 allein konnte ein leichte Verkürzung der Wachstumsfuge beobachtet werden, wobei prähypertrophe und hypertrophe Zone gleichermaßen betroffen waren. Dieser Phänotyp war verstärkt in Mäusen mit zusätzlichem heterozygotem Smad5-Hintergrund. Eine Verringerung der Proliferationsrate konnte zusammen mit einer verminderten Ihh-Expression nachgewiesen werden. Zusätzlich konnte anhand von Röntgenaufnahmen eine Dysorganisation der nasalen Region und ein fehlendes nasales Septum beobachtet werden. Produktion und Mineralisation der extrazellulären Matrix waren nicht beeinträchtigt. Um die Rolle der BMP- und TGFbeta-Signalkaskaden während der endochondralen Ossifikation zu vergleichen, wurden transgene Mäuse generiert, in denen die TGFbeta-Signalkaskade spezifisch in proliferierenden Chondrozyten gestört war. Zwei Mauslinien, die ähnliche Phänotypen zeigten, wurden untersucht. Esl1 ist ein TGFbeta-bindendes Protein, von dem man annimmt, dass es die TGFbeta-Signalkaskade inhibieren kann. Esl1-knock out-Mäuse sind kleiner als Wildtypmäuse und die Überexpression von Esl1 in proliferierenden Chondrozyten führt zu einer Verlängerung der Wachstumsfuge und einer verstärkten Proliferationsrate. Knorpelmarker, wie Col2a1 und Sox9 sind in diesen Mäusen herunterreguliert, während Col10a1 und Ihh als Marker für die hypertrophe und prähypertrophe Zone herunterreguliert waren. Dies führt zu der Annahme, dass mehr Zellen in die terminale Differenzierung eintreten. Bei transgenen Mäusen, in denen ein dominant-negativer (dn) TGFbeta-Rezeptor in proliferierenden Chondrozyten überexprimiert wurde, konnte eine verlängerte prähypertrophe Zone, eine erhöhte Ihh-Expression, sowie eine verstärkte Proliferationsrate beobachtet werden. Zusätzlich konnte in homozygoten Tieren ein craniofacialer Phänotyp beschrieben werden, der zu Problemen bei der Nahrungsaufnahme und damit zu einer starken Wachstumsbeeinträchtigung führte. Die BMP- und TGFbeta-Signalkaskaden haben möglicherweise antagonistische Effekte in der Wachstumsfuge. Während der Ausfall von BMP in proliferierenden Chondrozyten aufgrund einer gesunkenen Proliferationsrate zu einer Verkürzung der Wachstumsfuge führte, kann man in Mäusen mit einer Störung der TGFbeta-Signalkaskade eine verstärkte Proliferation in einer daher verlängerten Wachstumsfuge beobachten. Ein weiteres Ziel dieser Arbeit war die Generation einer transgenen Mauslinie, die die Cre-Rekombinase spezifisch in hypertrophen Chondrozyten exprimiert. Promoterstudien mit transgenen Mäusen weisen darauf hin, dass ein putatives AP1-Element, etwa 4 kb vor dem ersten Exon des Col10a1 gelegen, wichtig für die spezifische Expression in hypertrophen Chondrozyten ist. Ein Konstrukt, dass vier Kopien dieses Elements und den basalen Promoter enthält, wurde benutzt, um die Cre-Rekombinase spezifisch zu exprimieren. Diese Mauslinie befindet sich in der Testphase und erste Daten deuten auf eine spezifische Expression der Cre-Rekombinase in hypertrophen Chondrozyten hin.

Periosteal BMP2 activity drives bone graft healing

Bone graft incorporation depends on the orchestrated activation of numerous growth factors and cytokines in both the host and the graft. Prominent in this signaling cascade is BMP2. Although BMP2 is dispensable for bone formation, it is required for the initiation of bone repair; thus understanding the cellular mechanisms underlying bone regeneration driven by BMP2 is essential for improving bone graft therapies. In the present study, we assessed the role of Bmp2 in bone graft incorporation using mice in which Bmp2 has been removed from the limb prior to skeletal formation (Bmp2(cKO)). When autograft transplantations were performed in Bmp2cKO mice, callus formation and bone healing were absent. Transplantation of either a vital wild type (WT) bone graft into a Bmp2(cKO) host or a vital Bmp2(cKO) graft into a WT host also resulted in the inhibition of bone graft incorporation. Histological analyses of these transplants show that in the absence of BMP2, periosteal progenitors remain quiescent and healing is not initiated. When we analyzed the expression of Sox9, a marker of chondrogenesis, on the graft surface, we found it significantly reduced when BMP2 was absent in either the graft itself or the host, suggesting that local BMP2 levels drive periosteal cell condensation and subsequent callus cell differentiation. The lack of integrated healing in the absence of BMP2 was not due to the inability of periosteal cells to respond to BMP2. Healing was achieved when grafts were pre-soaked in rhBMP2 protein, indicating that periosteal progenitors remain responsive in the absence of BMP2. In contrast to the requirement for BMP2 in periosteal progenitor activation in vital bone grafts, we found that bone matrix-derived BMP2 does not significantly enhance bone graft incorporation. Taken together, our data show that BMP2 signaling is not essential for the maintenance of periosteal progenitors, but is required for the activation of these progenitors and their subsequent differentiation along the osteo-chondrogenic pathway. These results indicate that BMP2 will be among the signaling molecules whose presence will determine success or failure of new bone graft strategies.

Chondrogenic potential of human synovial mesenchymal stem cells in alginate

OBJECTIVE: In a recent study, we demonstrated that mesenchymal stem cells (MSCs) derived from the synovial membranes of bovine shoulder joints could differentiate into chondrocytes when cultured in alginate. The purpose of the present study was to establish the conditions under which synovial MSCs derived from aging human donors can be induced to undergo chondrogenic differentiation using the same alginate system. METHODS: MSCs were obtained by digesting the knee-joint synovial membranes of osteoarthritic human donors (aged 59-76 years), and expanded in monolayer cultures. The cells were then seeded at a numerical density of 4x10(6)/ml within discs of 2% alginate, which were cultured in serum-containing or serum-free medium (the latter being supplemented with 1% insulin, transferrin, selenium (ITS). The chondrogenic differentiation capacity of the cells was tested by exposing them to the morphogens transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and BMP-7, as well as to the synthetic glucocorticoid dexamethasone. The relative mRNA levels of collagen types I and II, of aggrecan and of Sox9 were determined quantitatively by the real-time polymerase chain reaction (PCR). The extracellular deposition of proteoglycans was evaluated histologically after staining with Toluidine Blue, and that of type-II collagen by immunohistochemistry. RESULTS: BMP-2 induced the chondrogenic differentiation of human synovial MSCs in a dose-dependent manner. The response elicited by BMP-7 was comparable. Both of these agents were more potent than TGF-beta1. A higher level of BMP-2-induced chondrogenic differentiation was achieved in the absence than in the presence of serum. In the presence of dexamethasone, the BMP-2-induced expression of mRNAs for aggrecan and type-II collagen was suppressed; the weaker TGF-beta1-induced expression of these chondrogenic markers was not obviously affected. CONCLUSIONS: We have demonstrated that synovial MSCs derived from the knee joints of aging human donors possess chondrogenic potential. Under serum-free culturing conditions and in the absence of dexamethasone, BMP-2 and BMP-7 were the most potent inducers of this transformation process.

Expression of cartilage-related genes in bovine synovial tissue

The synovium contains mesenchymal stem cells with chondrogenic potential. Although synovial and articular cartilage tissue develop from a common pool of mesenchymal cells, little is known about their genetic commonalities. In the present study, the mRNA levels for several cartilage-related proteins, namely, cartilage oligomeric matrix protein (COMP), Sox9, aggrecan, and collagen types I, II, IX, X, and XI, were measured using the real-time polymerase chain reaction. Our data reveal the synovium of calf metacarpal joints to physiologically express not only type I collagen but also COMP, Sox9, aggrecan, and collagen types X and XI. The mRNA levels for the latter five proteins lie between 2% and 15% of those in articular cartilage. We speculate that these genes are being expressed by chondroprogenitor cells, whose presence in the synovium reflects a common ontogenetic phase in the fetal development of this tissue and of articular cartilage.