Human Hedgehog-interacting Protein Research Articles

Circulating HHIP Levels in Women with Insulin Resistance and PCOS: Effects of Physical Activity, Cold Stimulation and Anti-Diabetic Drug Therapy.

Serum human hedgehog-interacting protein (HHIP) concentration is associated with diabetes. However, the relationship between HHIP and polycystic ovary syndrome (PCOS) or abnormal sex hormones remains unknown. This study was an observational cross-sectional study, with additional short-term intervention studies and follow-up studies. Bioinformatics analysis was performed to explore the association of PCOS with metabolic-related genes and signaling pathways. OGTT and EHC were performed on all participants. Lipid infusion, cold exposure, and 45-min treadmill test were performed on all healthy women. A total of 137 women with PCOS were treated with metformin, GLP-1RA, or TZDs for 24 weeks. Serum HHIP levels were higher in insulin resistance (IR) and PCOS women. Circulating HHIP levels were significantly correlated with adiponectin (Adipoq) levels, obesity, IR, and metabolic indicators. A correlation presented between HHIP and DHEA-S, FAI, SHBG, and FSH. Serum HHIP levels were significantly elevated by oral glucose challenge in healthy women, but not affected by EHC. Lipid infusion decreased serum HHIP levels, while cold exposure increased HHIP levels in healthy women. GLP-1RA and TZD treatment reduced serum HHIP levels in PCOS women, while metformin treatment did not affect HHIP levels. HHIP may be a useful biomarker and novel drug target for PCOS and IR individuals.

MicroRNA-221 promotes tumor progression by targeting HHIP in human glioblastoma.

BackgroundMicroRNAs are found to be aberrantly expressed in multiple cancers, including glioblastoma (GBM), and microRNA-221 (miR-221) has been verified as an oncogene in various human cancers. Nevertheless, the role of miR-221 in GBM is unclear. This study aimed to investigate the miR-221 expression level in GBM and to evaluate its function and underlying mechanisms.MethodsWestern blotting and qPCR were used to determine the expression of human hedgehog-interacting protein (HHIP) and miR-221 levels. MiR-221-inhibited cell models were constructed, and siRNA was used for HHIP silencing. Cell proliferation was analyzed by MTT and colony formation assays and a subcutaneous xenograft model. Cell migration and invasion was analyzed by wound healing and Transwell invasion assays. A dual luciferase reporter assay system was used to clarify the relationship between miR-221 and HHIP.ResultsThe results of this study revealed that miR-221 expression was upregulated in GBM tissues and A172, U251, as well as T98G cells, as detected by real-time PCR analysis. MTT, Transwell, and colony formation assays revealed that miR-221 knockdown could suppress GBM cells from proliferating, migrating, and invading in vitro. Moreover, animal experiments showed that tumor growth in vivo was inhibited when miR-221 expression decreased. Furthermore, HHIP was predicted and verified to be a target of miR-221 by bioinformatics analysis, and luciferase and western blot assays. In addition, HHIP silencing rescued the suppressive effect of a miR-221 inhibitor on the proliferation, migration, and invasion of GBM cells.ConclusionsOur results indicated that miR-221 is upregulated in GBM and enhances tumor progression by targeting HHIP, which suggests this may be a potential therapeutic target for GBM.

HHIP Overexpression Suppresses Human Gastric Cancer Progression and Metastasis by Reducing Its CpG Island Methylation.

Human hedgehog-interacting protein (HHIP), a negative regulator of hedgehog (HH) signaling pathway, has been reported to be dysregulated in many types of cancer, including gastric cancer. However, the inhibitory role of HHIP as well as the underlying molecular mechanism of HHIP regulation in gastric cancer haven’t been fully elucidated yet. In this study, we demonstrated that HHIP overexpression significantly suppressed the proliferation and invasion of AGS cells evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and transwell assays, respectively. Interestingly, methylation-specific polymerase chain reaction (MS-PCR, MSP) showed that HHIP overexpression dramatically decreased its de novo promoter methylation levels in AGS cells. Furthermore, HHIP expression was higher in adjacent non-cancerous tissue compared to matched gastric cancer tissue. High HHIP level was negatively correlated with metastasis (p = 0.035) but not local recurrence (p = 0.58). Taken together, our study suggested that HHIP can modulate gastric cancer progression and metastasis via regulation of its de novo promoter methylation levels in a feedback manner. Lower HHIP levels is positively associated with gastric cancer metastasis, which not only indicates HHIP could be served as a protective marker for gastric cancer, but also suggests restoring HHIP expression might be a potential therapeutic strategy for clinical treatment.

Inhibition of HHIP Promoter Methylation Suppresses Human Gastric Cancer Cell Proliferation and Migration

Background/Aims: Human hedgehog-interacting protein (HHIP) is a negative regulator of the hedgehog (HH) signaling pathway. It is deregulated in gastric cancer. The underlying molecular mechanism of HHIP-induced inhibition of HH signaling remains to be determined. Methods: A lentiviral HHIP expression vector (“LV-HHIP”) was established to exogenously over-express HHIP in gastric cancer cells. HHIP protein and mRNA were tested by Western blotting assay and quantitative real-time PCR assay, respectively. Cell survival was tested by the Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was examined by the BrdU ELISA assay and [H³] Thymidine DNA incorporation assay. Cell invasion and migration were tested by the phagokinetic track assay and the “Transwell” assay. The bisulfite-sequencing PCR was applied to test HHIP promoter methylation. Results: In the established (AGS cell line) and primary human gastric cancer cells, LV-HHIP transfection increased HHIP expression and inhibited cancer cell survival and proliferation as well as cell migration and invasion. Furthermore, LV-HHIP significantly attenuated promoter methylation of the endogenous HHIP gene in AGS cells, causing it upregulation. Inhibition of methylation by 5-aza-dc similarly induced HHIP expression in gastric cancer cells, which inhibited cancer cell proliferation and migration. Conclusions: Our results suggest that inhibition of HHIP promoter methylation can efficiently inhibit human gastric cancer cell proliferation and migration.

Gene susceptibility identification in a longitudinal study confirms new loci in the development of chronic obstructive pulmonary disease and influences lung function decline.

BackgroundTo identify COPD associated gene susceptibility and lung function in a longitudinal cohort including COPD and subjects who were at risk for developing COPD, and to replicate this in two cross-sectional and longitudinal populations in Chinese Han population.MethodsThree cohorts were recruited in this study, including an 18-year follow-up population (306 COPD and 743 control subjects) in one village in 1992 and it changed to 409 COPD and 611 controls in 2010, a 2 year follow-up study in another village (374 COPD and 377 controls) and another 2 year follow-up one in a city (541 COPD and 560 controls) in 2010. Sixteen candidate single nucleotide polymorphisms (SNPs) were selected for genotyping. Among them, 5SNPs in or near HHIP, 1SNP in IREB2 and 1SNP in FAM13A were previously reported to be associated with COPD susceptibility or lung function decline. And another 9SNPs were selected from HapMap website as HHIP tags. In 2010, totaling 1,324 COPD patients and 1,548 healthy controls were finally included in our genetic susceptibility analyses.ResultsWe identified two new regions showing an association with COPD susceptibility in the Human Hedgehog interacting protein (HHIP) rs11100865 and rs7654947, and we confirmed that the family with sequence similarity 13 member A gene (FAM13A) rs7671167 was associated with the development of COPD in Chinese Han population. And the HHIP rs7654947 and FAM13A rs7671167 were associated with lung function decline, and this result was replicated in other two populations.ConclusionsThese results suggest an important role of the HHIP and FAM13A regions as genetic risk factors for COPD development and lung function decline in Chinese Han population. Future research on these genes should focus on the molecular mechanisms of these genes on developing COPD and creating therapies to alleviate reduced lung function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0209-3) contains supplementary material, which is available to authorized users.

Occurrence of HHIP gene CpG island methylation in gastric cancer.

The present study aimed to observe the methylation status of the CpG islands at the human hedgehog interacting protein (HHIP) gene in gastric cancer tissues, peritumoral tissues and the AGS cell line, to analyze the association between the methylation status of the CpG islands and the tumorigenesis of gastric cancer. The HHIP mRNA expression in 60 human gastric carcinnoma tissues, peritumoral tissues and the gastric carcinoma AGS cell line were detected by reverse transcription polymerase chain reaction (RT-PCR). The HHIP methylation status of the promoter region in the gastric carcinnoma tissues and peritumoral tissues was detected by methylation-specific PCR (MSP). Prior to and following treatment with methyl transferase inhibitor 5-aza-2′-deoxycitydine (5-aza-dc), the HHIP mRNA expression level, the methylation status of the promoter region and the methylation site loci on the CpG islands in the AGS cells were detected by RT-PCR, MSP and bisulfite sequencing PCR (BSP), respectively. The correlation between the methylation status of the CpG islands at the HHIP promoter region and the HHIP mRNA expression level were analyzed. It was found that the expression level of the HHIP mRNA in the gastric carcinoma tissues was significantly lower than that in the adjacent tissues (0.82±0.38 vs. 1.60±0.26, respectively; P<0.001). No significant correlations were observed between the expression of HHIP mRNA and age, gender, tumor-node-metastasis stage, differentiation degree and presence of lymph node metastasis (P>0.05). The degree of methylation of the HHIP gene promotor in the peritumoral tissues (17.7±3.59%) was significantly lower than that in the gastric cancer tissues (62.9±6.14%) and in the AGS cells (99.7±0.67%) (P<0.05). Compared with prior to 5-aza-dc intervention, the HHIP mRNA expression level in the AGS cells was significantly increased subsequent to intervention (0.21±0.12 vs. 4.68±0.22; P<0.01), while the degree of methylation in the AGS cells was significantly decreased (90.2±0.67 vs. 10.1±0.21%; P<0.01), and the methylation sites in CpG islands were significantly reduced. The degree of HHIP methylation showed a negative correlation with the level of mRNA expression (r=−0.693; P<0.01). It can be hypothesized that a high degree of methylation of the HHIP gene promoter CpG islands in gastric cancer tissues and cells causes a decrease in HHIP mRNA expression, which may be involved in the carcinogenesis of gastric cancer.

Altered expression of PTCH and HHIP in gastric cancer through their gene promoter methylation: Novel targets for gastric cancer

Human hedgehog-interacting protein (HHIP) and protein patched homolog (PTCH) are two negative regulators of the hedgehog signal, however, the mechanism of action in gastric cancer is unknown. Methylation of TSG promoters has been considered as a causative mechanism of tumorigenesis. In the present study, we first determined the expression of PTCH and HHIP mRNA and protein in gastric cancer tissues and adjacent normal tissues, and then detected methylation of the two genes to associate their expression and gene promoter methylation in gastric cancer. Expression in gastric cancer tissues and the cancer cells (AGS) were evaluated by reverse transcription-PCR (RT-PCR), qRT-PCR and IHC, while the methylation expression was valued by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). Cell viability and apoptosis were analyzed by MTT assay and flow cytometry following treatment with 5-aza-dc. Results showed that PTCH and HHIP expression was reduced in gastric cancer tissues that were not associated with clinical features. Moreover, methylation of the promoters was reversely correlated with the expression. Following treatment with 5-aza-dc, AGS reduced cell viability and induced apoptosis, which is associated with upregulation of HHIP expression. The data demonstrated that loss of expression of HHIP and PTCH is associated with the methylation of gene promoters. In addition, 5-aza-dc-induced apoptosis correlated with the upregulation of HHIP expression in AGS. The findings demonstrated that the PTCH and HHIP genes may be novel targets for the control of gastric cancer.

Stromal Cells Expressing Hedgehog-Interacting Protein Are One of the Main Regulators of the Hedgehog Signaling System in Myeloid Neoplasms

Abstract 3840Aberrant reactivation of Hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasm is unclear. In this study we assessed the expression of Hh-related proteins in normal human CD34+ cells, CD34+ leukemic/dysplastic cells and BM stromal cells. Both Indian hedgehog (Ihh) and its signal transducer, SMO, were expressed in CD34+ acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) derived cells, suggesting that Ihh could be affected in an autocrine manner. Remarkably, expression of the endogenous Hh signaling inhibitor, human hedgehog-interacting protein (HHIP), in AML/MDS-associated stromal cells was significantly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression level in BM stromal cells highly correlated with their tumor-supporting activity of SMO+ leukemic cells. Demethylating agent 5-aza-dC rescued HHIP expression via demethylation of HHIP gene and reduced the leukemia-supporting activity of AML/MDS-associated stromal cells. This effect of 5-aza-dC was negated by HHIP shRNA transfer into stromal cells. These results indicate that suppression of stromal HHIP expression could be involved in the progression of AML/MDS and 5-aza-dC may improve the protective function of BM stromal cells for AML/MDS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signaling leads to disease. Multiple cell surface receptors are responsible for transducing and/or regulating Hh signals. Among these, the hedgehog-interacting protein (HIP) is a highly conserved, vertebrate-specific, inhibitor of Hh signaling. We have solved a series of crystal structures for the human HIP ectodomain and Desert Hh in isolation, as well as Sonic and Desert Hh-HIP complexes, with and without calcium. The interaction determinants, confirmed by biophysical studies and mutagenesis, reveal novel and distinct functions for Hh zinc- and calcium-binding sites; functions which appear common to all vertebrate Hhs. Zinc makes a key contribution to the Hh-HIP interface while calcium prevents electrostatic repulsion between the two proteins, thus playing a major modulatory role. This interplay of several metal-binding sites suggests a tuneable mechanism for regulation of Hh signaling.

Aberrant methylation of the human hedgehog interacting protein (HHIP) gene in pancreatic neoplasms

Hedgehog pathway overactivity has been implicated in the development of a variety of human cancers. The Human Hedgehog interacting protein (HHIP), a negative regulator of hedgehog signaling, has been shown to be underexpressed in pancreatic cancers. In this study we determined if the HHIP gene is a target for genetic and epigenetic alterations. While no mutations of HHIP were identified, we found complete methylation of the HHIP promoter CpG island in 3 pancreatic cancer cell lines, and partial hypermethylation in 13/17 (80%) pancreatic cancer cell lines, 35/75 (46%) primary pancreatic cancers and 14/18 (78%) pancreatic cancer xenografts, but no methylation in 13 normal pancreata. In pancreatic cancer cell lines, complete methylation was associated with absent or reduced HHIP expression by real-time RT-PCR. HHIP expression could be restored in methylated cell lines using epigenetic modifier drugs. Restoring the expression of HHIP in pancreatic cancer cells by 5-aza-2'-deoxycytidine led to a decrease in Gli reporter activity, consistent with downregulation of Hedgehog signaling. These results indicate in some pancreatic adenocarcinomas that HHIP is epigenetically inactivated by promoter methylation, and its silencing could contribute to the increased Hedgehog signaling observed in pancreatic neoplasms.

Localization of the human hedgehog‐interacting protein (Hip) in the normal and diseased pancreas

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis. Previously, it has been shown that Indian hedgehog (Ihh) and its two signaling receptors patched (Ptc) and smoothened (Smo) are involved in the pathogenesis of chronic pancreatitis (CP) and PDAC. In the current study we analyzed the expression, distribution, and function of another component of this signaling pathway, the human hedgehog-interacting protein (Hip), in the normal pancreas, CP and PDAC utilizing real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR), immunohistochemistry, immunofluorescence, Hip siRNA transfection, cell growth assays, and cell cycle analysis. By QRT-PCR, Hip mRNA levels were fifteenfold and fourteenfold increased in CP (n = 22) and PDAC (n = 31) tissues, respectively, compared to normal pancreatic tissues (n=20) and correlated with glioma associated antigen (Gli1) but not Ptc or Protein kinase A (PKA) mRNA levels. Only SU-8686 and BxPC-3 pancreatic cancer cells expressed Hip mRNA, whereas expression was below the level of detection in the other six pancreatic cancer cell lines tested. As shown by immunohistochemistry, Hip was expressed in normal pancreatic tissues mainly in the cytoplasm of islet cells and in smooth muscle cells of blood vessels. In contrast, in CP and PDAC there was a different distribution and staining intensity within the islets. Moreover, Hip immunoreactivity was observed in the tubular complexes, PanIN 1-3 lesions, as well as in pancreatic cancer cells. Incubation of pancreatic cancer cell lines with recombinant Hip revealed a growth inhibitory effect in SU-8686 and Capan-1 pancreatic cancer cells and no effect on cell growth in the other tested cell lines. In addition, silencing of Hip expression using specific siRNA molecules increased the growth of SU-8686 cells. In conclusion, Hip is expressed in the normal pancreas, CP and PDAC tissues. The different pattern of Hip expression and abnormal localization in the diseased pancreas suggest that the enhanced activation of hedgehog signaling in CP and PDAC is-at least in part-due to the aberrant responsiveness and expression of Hip in these diseases.

The human hedgehog-interacting protein gene: Structure and chromosome mapping to 4q31.21→q31.3

Hedgehog-interacting protein (Hhip) is a novel regulatory component in the vertebrate hedgehog-signalling pathway. The murine Hhip encodes a type I TM protein that attenuates hedgehog signalling by binding all three mammalian hedgehog proteins. Here we describe the cloning and characterisation of the homologous human hedgehog-interacting protein gene (HHIP). HHIP comprises 13 exons and spans >91kb encoding a protein of 700 aa which shares 94% sequence iden- tity with mouse Hhip. HHIP maps to chromosome 4q31.21→ q31.3. Additionally, we have mapped murine Hhip to chromosome 8.

Determination of the chromosomal location and genomic structure of the Hedgehog-Interacting Protein gene, and analysis of its role in Holoprosencephaly

Hedgehog-Interacting Protein (HIP) is a novel component of the Sonic Hedgehog (SHH) signaling pathway. Recently, defects in this pathway have been shown to cause holoprosencephaly (HPE), which is the most common birth defect of the brain and face in humans. In animal models knockout mice with homozygous null mutations for Shh displayed abnormalities consistent with HPE. Hip has been shown to function as a co-receptor and attenuate Hedgehog signaling by cell-surface binding of the Hedgehog protein and is hypothesized to act as part of a negative regulatory feedback loop. Although Hip is an important factor in the Shh signaling pathway, its potential role in HPE had not been examined in humans. Here, we report the complete gene structure of the human HIP gene and present its mutational analysis in HPE patients. No mutations were found either in the entire coding region or 1 kb upstream of the transcriptional start site (representing the putative promotor) suggesting this gene may not be involved in HPE pathogenesis.