Hedgehog signaling regulates differentiation from double-negative to double-positive thymocyte

The hedgehog (Hh) signaling pathway is involved in the development of many tissues. Here we show that sonic hedgehog (Shh) is involved in thymocyte development. Our data suggest that termination of Hh signaling is necessary for differentiation from CD4-CD8-double-negative (DN) to CD4+CD8+ double-positive (DP) thymocyte. Shh is produced by the thymic stroma, and Patched and Smoothened (Smo), the transmembrane receptors for Shh, are expressed in DN thymocytes. A neutralizing monoclonal antibody against Shh increases differentiation of DN to DP thymocytes, and Shh protein arrests thymocyte differentiation at the CD25+ DN stage, after T cell receptor beta (TCRbeta) gene rearrangement. We show that one consequence of pre-TCR signaling is downregulation of Smo, allowing DN thymocytes to proliferate and differentiate.     

Hedgehog signaling regulates proliferation of prostate cancer cells via stathmin1

Hedgehog (Hh) signaling is an essential pathway in embryonic development of prostate. Hh also plays roles in the proliferation of progenitor cells and cancer cells of adult prostate. However, how Hh signaling contributes to carcinogenesis of prostate is poorly understood. Stathmin1 is a microtubule-regulating protein that plays an important role in the assembly and disassembly of the mitotic spindle. Stathmin1 is expressed in normal developing mouse prostate and in prostate cancer. The expression pattern of stathmin1 is similar to that of Shh in prostate development and cancer, suggesting a connection between these two proteins. In this study, we examined the relationship between stathmin1 and Hh signaling. Here, we show that stathmin1 expression is regulated by Hh signaling in prostate cancer cells. Cyclopamine, a specific inhibitor of Hh signaling, reduced the expression of stathmin1 in prostate cancer cells. However, the Shh peptide induced stathmin1 expression. Overexpression of Gli1 further confirmed the relationship. Co-expression of stathmin1 and Patched 1, a receptor for Hh signaling was observed in prostate cancer tissues. Cyclopamine and stathmin1 siRNA both decreased proliferation of prostate cancer cells but did not produce an additive effect, suggesting a common pathway. These results suggest that Hh signaling regulates proliferation of prostate cancer cells by controlling stathmin1 expression.     

Hedgehog信号通路蛋白在肝内胆管细胞癌中的表达

目的 研究Hedgehog(Hh)信号通路相关蛋白(Gli 1,Ptch-1和Shh)在肝内胆管细胞癌(intrahepatic cholangio carcinoma,ICC)中的表达及意义.通过对ICC和癌旁组织中Hh通路相关蛋白(Gli 1、Ptch-1和Shh)的表达进行检测,探讨其与ICC临床病理特征之间的关系及意义.方法 免疫组化法对43例ICC组织及癌旁组织进行Gli 1、Ptch-1和Shh蛋白的检测;Real-time RT-PCR检测12例ICC组织和癌旁组织中mRNA的表达,并与患者临床病理特征进行相关性分析.结果 43例ICC组织中,Gli 1、Ptch-1和Shh阳性表达率分别为74.4%(32/43)、69.8% (30/43),和62.8%(27/43),与癌旁组织表达比较差异具有统计学意义(P值分别为0.003、0.006和0.008);三者的表达与肿瘤的大小有关(P值分别为0.039、0.044和0.047);三者的表达与肿瘤的分化程度相关(P值分别为0.026,0.039和0.049);三者的表达与淋巴结转移具有相关性(P均<0.05).Real-time PCR结果显示,12例ICC组织中均有Gli 1、Ptch-1和Shh的表达,且基因表达水平均高于癌旁正常组织(P均=0.001).结论 Hedgehog信号通路的相关蛋白在ICC组织中高表达,其异常激活可能与肝内胆管细胞癌发生密切相关.Gli 1、Ptch-1和Shh的表达可能与肿瘤的生长和转移密切相关.     

Hedgehog信号通路阻断剂环巴胺体外对佐剂性关节炎大鼠的关节软骨细胞增殖的影响

目的探讨Hedgehog(Hh)信号通路抑制剂环巴胺体外对佐剂性关节炎大鼠(AA)模型的关节软骨细胞增殖的影响和部分机制。方法弗氏完全佐剂诱导AA大鼠,测量关节炎指数和继发性足肿胀度,HE染色观察两组软骨组织生长情况;取AA大鼠踝关节软骨组织,采用胰蛋白酶-胶原酶法分离、培养、鉴定,环巴胺(0、0.05、0.5、5、20μmol/L)体外给药,MTT法检测AA大鼠踝关节软骨细胞增殖,Annexin V-FITC/PI双染检测AA大鼠踝关节软骨细胞凋亡,Western blotting检测AA大鼠踝关节软骨细胞Shh、Ptch1、Gli1的蛋白表达。结果弗氏完全佐剂诱导后,与正常大鼠相比,AA大鼠关节炎指数和继发性足肿胀度明显升高,HE染色显示,AA大鼠踝关节软骨组织有破坏;甲苯胺蓝和Ⅱ型胶原鉴定体外成功培养AA大鼠踝关节软骨细胞;体外给药环巴胺(0.05、0.5、5、20μmol/L)可升高AA模型关节软骨细胞增殖,流式细胞检测结果显示,环巴胺能降低AA模型软骨细胞凋亡率;与未用环巴胺组相比,环巴胺(0.5、5、20μmol/L)给药对AA软骨细胞中Hh信号通路相关蛋白(Shh、Ptch1、Gli1)表达显著下降。结论弗氏完全佐剂诱导建立AA大鼠模型成立,体外给药环巴胺可抑制AA大鼠软骨细胞的增殖,抑制软骨细胞的凋亡,该作用与抑制AA大鼠软骨细胞Hh信号有关。     

Hedgehog signaling is involved in differentiation of normal colonic tissue rather than in tumor proliferation

The Hedgehog (Hh) pathway is a main regulation cascade in embryonic differentiation. It is also present in adult tissues and unusual expression has been associated with formation of benign and malignant lesions. We examined the presence of the Hedgehog pathway in normal and pathological human colon tissue. Components investigated include Sonic (Shh), Indian (Ihh), and Desert Hedgehog (Dhh), Gli1, Gli2, Gli3, and Patched (Ptch). Pathological tissue samples comprised 23 benign and 20 malignant lesions of human colon. The influence of the Hedgehog pathway on differentiation and proliferation has been investigated by analyzing the effect of the pathway inhibitor Cyclopamine on human colon cancer cell lines HT29 and CaCo2. In normal colon, we detected expression of Shh and Dhh within the lining epithelium and Patched, Gli1, and Gli2 along the whole crypts. Within all benign lesions, positive staining of Shh, Dhh, Gli1, Gli2, and Ptch was detected. Expression of Shh and Dhh was restricted to single cell aggregates. Malignant lesions also displayed focal staining pattern for Shh and Dhh but to a much lesser extent. We conclude that Hedgehog signaling is involved rather in constant differentiation and renewing of the colonic lining epithelium than in cancer formation, growth, or proliferation.     

A direct requirement for Hedgehog signaling for normal specification of all ventral progenitor domains in the presumptive mammalian spinal cord

The hedgehog signaling pathway organizes the developing ventral neural tube by establishing distinct neural progenitor fates along the dorsoventral axis. Smoothened (Smo) is essential for all Hedgehog (Hh) signaling, and genetic inactivation of Smo cells autonomously blocks the ability of cells to transduce the Hh signal. Using a chimeric approach, we examined the behavior of Smo null mutant neural progenitor cells in the developing vertebrate spinal cord, and we show that direct Hh signaling is essential for the specification of all ventral progenitor populations. Further, Hh signaling extends into the dorsal half of the spinal cord including the intermediate Dbx expression domain. Surprisingly, in the absence of Sonic hedgehog (Shh), we observe the presence of a Smo-dependent Hh signaling activity operating in the ventral half of the spinal cord that most likely reflects Indian hedgehog (Ihh) signaling originating from the underlying gut endoderm. Comparative studies of Shh, Smo, and Gli3 single and compound mutants reveal that Hh signaling acts in part to specify neural cell identity by counteracting the repressive action of Gli3 on p0, p1, p2, and pMN formation. However, whereas these cell identities are restored in Gli3/Smo compound mutants, correct stratification of the rescued ventral cell types is lost. Thus, Hh signaling is essential for organizing ventral cell pattern, possibly through the control of differential cell affinities.     

Hedgehog/Gli1通路在雷奈酸锶促进骨髓间充质干细胞成骨分化过程中的作用

目的:探讨Hedgehog/Gli1信号通路在雷奈酸锶(strontium ranelate,Sr)促进骨髓间充质干细胞(BMSCs)向成骨细胞分化中的作用。方法:体外分离培养大鼠BMSCs,诱导其成骨分化,根据实验目的加入不同浓度的Sr、Hedgehog受体拮抗剂cyclopamine(Cy)及Gli1小干扰RNA(Gli1-siRNA)。用Western blotting法检测Gli1及Runx2的表达,酶标法检测碱性磷酸酶(ALP)活性,茜素红染色法检测钙结节水平。结果:应用不同浓度的Sr(0.1~5 mmol/L)处理BMSCs细胞7 d后,细胞内Gli1蛋白表达增高,Sr的浓度为3 mmol/L时,Gli1表达达到高峰;使用Cy与Sr共处理BMSCs 7 d,能拮抗Sr对Gli1蛋白表达的上调作用;应用Gli1-siRNA转染细胞后,能下调Gli1蛋白的表达,并抑制Sr对Gli1下游Runx2蛋白表达的上调作用,还可拮抗Sr对ALP活性及钙化结节形成的促进作用。结论:Hedgehog/Gli1通路参与了Sr促进骨髓间充质干细胞向成骨分化的过程。     

Commercially available recombinant sonic hedgehog up-regulates Ptc and modulates the cytokine and chemokine expression of human macrophages: an effect mediated by endotoxin contamination?

The Sonic hedgehog (Shh) signalling pathway plays an important role in developmental patterning and proliferation. Recent evidence suggests that Shh also plays a role in the development of the immune system. Here, we demonstrate that components of the Shh signalling pathway are expressed in human macrophages and that the receptor for Shh, Ptc, is up-regulated by a commercially available recombinant preparation of Shh (CArShh). Further, we report that the addition of CArShh up-regulates the production of IL-6, IL-8, MCP-1, IP-10, MIG and RANTES by macrophages, an effect enhanced by the presence of fetal calf serum in the culture medium. In contrast, TGF-beta, TNF-alpha, IL-1b, IL-12 and IL-10 production were not modulated by CArShh and VEGF was minimally up-regulated even in the presence of serum. The up-regulation of these cytokines and chemokines was abrogated by CD14 inhibition and polymixin B, but not reliably inhibited by the specific Shh pathway inhibitor cyclopamine. These results suggest that, although components of the Shh signalling pathway are expressed in macrophages, the modulation of macrophage cytokine and chemokine effector function seen in response to commercially available rShh results from low levels of endotoxin contained within the CArShh preparations employed to explore the effects of Shh in vitro.     

Meckel's cartilage differentiation is dependent on hedgehog signaling

The hedgehog (Hh) signaling pathway has been shown to be essential for craniofacial development. Although mandibular arch derivatives are largely absent in Shh null mice, little is known about the role of Hh signaling during Meckel's cartilage development per se. Mandible development is dependent on the morphogenesis of Meckel's cartilage, which then serves as a template for subsequent skeletal differentiation. In this study, we examine the biological function of Hh signaling during Meckel's cartilage development in vivo and in vitro. E13.5 Shh null mice present a small mesenchymal condensation in the region of a presumptive Meckel's cartilage in the hypoplastic mandibular arch. By E15.5, the Shh mutant exhibits a mere remnant of the mandibular arch, without evidence of Meckel's cartilage differentiation. Further, wild-type embryonic (E11 or E12) mandibular explants cultured for up to 5 days in the presence of cyclopamine, a steroidal alkaloid that specifically disrupts the Hh signaling pathway, exhibit a stage-dependent inhibition of Meckel's cartilage chondroblast differentiation to mature chondrocytes. This phenotype can be rescued by exogenous FGF8, a downstream effector of Hh signaling. Taken together, our results indicate that the Hh signaling pathway is critical to Meckel's cartilage ontogenesis and the rate of chondrogenesis, but not to initial primordium formation. The reliance on Hh signaling is stage dependent.     

Expression of hedgehog pathway components in prostate carcinoma microenvironment: shifting the balance towards autocrine signalling

Tzelepi V, Karlou M, Wen S, Hoang A, Logothetis C, Troncoso P & Efstathiou E
(2011) Histopathology 58, 1037–1047
Expression of hedgehog pathway components in prostate carcinoma microenvironment: shifting the balance towards autocrine signalling Aims: The hedgehog (Hh) signalling pathway has been implicated in the pathogenesis and aggressiveness of prostate cancer through epithelial–mesenchymal interactions. The aim of this study was to elucidate the cell‐type partitioned expression of the Hh pathway biomarkers in the non‐neoplastic and tumour microenvironments and to correlate it with the grade and stage of prostate cancer. Methods and results: Expression of the Hh pathway components (Shh, Smo, Ptch, Gli1) in the microenvironment of non‐neoplastic peripheral zone (n = 119), hormone‐naive primary prostate carcinoma (n = 141) and castrate‐resistant bone marrow metastases (n = 53) was analysed using immunohistochemistry in tissue microarrays and bone marrow sections. Results showed that epithelial Shh, Smo and Ptch expression was up‐regulated, whereas stromal Smo, Ptch, and Gli1 expression was down‐regulated in prostate carcinomas compared to non‐neoplastic peripheral zone tissue. Ptch expression was modulated further in high‐grade and high‐stage primary tumours and in bone marrow metastases. Hh signalling correlated with ki67 and vascular endothelial growth factor (VEGF) but not with CD31 expression. Conclusion: Our results highlight the importance of Hh‐mediated epithelial–mesenchymal interactions in the non‐neoplastic prostate and imply that shifting the balance from paracrine towards autocrine signalling is important in the pathogenesis and progression of prostate carcinoma.     

Hedgehog信号通路抑制剂对肝内胆管癌RBE细胞生物学行为的影响

 目的: 研究Hedgehog(Hh)信号通路特异性抑制剂环杷明(cyclopamine)对人肝内胆管癌细胞株RBE生物学行为的影响。方法: 用台盼蓝染色计数法和MTT比色法检测环杷明对RBE细胞增殖的影响;流式细胞术检测凋亡率,Transwell检测环杷明处理前后RBE细胞侵袭能力的变化,Western blot检测环杷明处理前后RBE细胞中Gli1和MMP-9的蛋白表达变化。结果: 环杷明对RBE细胞株增殖的抑制作用呈剂量和时间依赖性。环杷明作用细胞24 h、48 h、72 h后,RBE细胞凋亡率逐渐升高,明显高于对照组的凋亡率。Transwell检测对照组穿透细胞数为154.52±13.61,而实验组穿透数为62.00±12.17,侵袭能力明显下降(P<0.01)。Gli1和MMP-9蛋白均在RBE细胞中表达,环杷明下调RBE细胞的Gli1和MMP-9表达。结论: 阻断Hh信号通路能抑制RBE细胞的增殖,促进其凋亡,并抑制其侵袭能力。