Control of the Hedgehog pathway by compartmentalized PKA in the primary cilium.

The Hedgehog (Hh) signaling is one of the essential signaling pathways during embryogenesis and in adults. Hh signal transduction relies on primary cilium, a specialized cell surface organelle viewed as the hub of cell signaling. Protein kinase A (PKA) has been recognized as a potent negative regulator of the Hh pathway, raising the question of how such a ubiquitous kinase specifically regulates one signaling pathway. We reviewed recent genetic, molecular and biochemical studies that have advanced our mechanistic understanding of PKA's role in Hh signaling in vertebrates, focusing on the compartmentalized PKA at the centrosome and in the primary cilium. We outlined the recently developed genetic and optical tools that can be harvested to study PKA activities during the course of Hh signal transduction.

SHH-N non-canonically sustains androgen receptor activity in androgen-independent prostate cancer cells.

Prostate cancer is the second most frequent cancer diagnosed in men worldwide. Localized disease can be successfully treated, but advanced cases are more problematic. After initial effectiveness of androgen deprivation therapy, resistance quickly occurs. Therefore, we aimed to investigate the role of Hedgehog-GLI (HH-GLI) signaling in sustaining androgen-independent growth of prostate cancer cells. We found various modes of HH-GLI signaling activation in prostate cancer cells depending on androgen availability. When androgen was not deprived, we found evidence of non-canonical SMO signaling through the SRC kinase. After short-term androgen deprivation canonical HH-GLI signaling was activated, but we found little evidence of canonical HH-GLI signaling activity in androgen-independent prostate cancer cells. We show that in androgen-independent cells the pathway ligand, SHH-N, non-canonically binds to the androgen receptor through its cholesterol modification. Inhibition of this interaction leads to androgen receptor signaling downregulation. This implies that SHH-N activates the androgen receptor and sustains androgen-independence. Targeting this interaction might prove to be a valuable strategy for advanced prostate cancer treatment. Also, other non-canonical aspects of this signaling pathway should be investigated in more detail and considered when developing potential therapies.

Cells responding to hedgehog signaling contribute to the theca of ovarian follicles.

Cell-fate mapping was used to identify cells that respond to the hedgehog (HH) signaling pathway and that are incorporated into the theca cell layer during ovarian follicle development. Expression of Gli1 is increased by HH signaling and can be used as a marker of cells responsive to HH in reporter mice. In transgenic Gli1ERcre/tdT mice, injection of tamoxifen (TAM) induces cre-mediated recombination and expression of td tomato (tdT) which leads to permanent fluorescent marking of cells expressing Gli1 and their progeny. The identity of tdT-positive cells was determined by co-staining ovaries for endothelial cells (CD31), pericytes (CSPG4), vascular smooth muscle cells (VSMC; smooth muscle actin) and steroidogenic cells (cytochrome P450 17A1). Gli1ERcre/tdT mice were injected with TAM on the day of birth. Cells positive for tdT in 2-day-old mice were identified as pericytes, located primarily in the medulla of the ovary in close proximity to endothelial cells. In both prepubertal mice and adult mice treated with equine chorionic gonadotropin to induce the formation of preovulatory follicles, tdT-positive cells were located within the theca cell layer and were identified as pericytes, VSMC and steroidogenic theca cells. Granulosa cells are known to express two HH ligands, Indian HH and desert HH (DHH). In DHHcre/tdT reporter mice, endothelial cells were marked as tdT-positive indicating that endothelial cells, in addition to granulosa cells, express Dhh in the ovary. These findings suggest that HH signaling may stimulate the development of the vasculature along with steroidogenic capacity of the theca layer during follicle development.

The inhibition of microRNA-326 by SP1/HDAC1 contributes to proliferation and metastasis of osteosarcoma through promoting SMO expression.

Osteosarcoma (OS) is a malignant bone cancer lacking of effective treatment target when the metastasis occurred. This study investigated the implication of MicroRNA-326 in OS proliferation and metastasis to provide the clue for the treatment of metastatic OS. This study knocked down SP1 in MG63 and 143B cells and then performed Microarray assay to find the expression of miRNAs that were influenced by SP1. MTT, EdU, wound-healing and cell invasion assays were performed to evaluated cell proliferation and invasion. OS metastasis to lung was detected in a nude mice model. ChIP assay and DAPA were applied to determine the regulatory effect of SP1 and histone deacetylase 1 (HDAC) complex on miR-326 expression. Human OS tissues showed lowly expressed miR-326 but highly expressed Sp1 and HDAC. Sp1 recruited HDAC1 to miR-326 gene promoter, which caused the histone deacetylation and subsequent transcriptional inhibition of miR-326 gene. miR-326 deficiency induced the stimulation of SMO/Hedgehog pathway and promoted the proliferation and invasion of 143B and MG63 cells as well as the growth and metastasis in nude mice. SP1/HDAC1 caused the transcriptional inhibition of miR-326 gene by promoting histone deacetylation; miR-326 deficiency conversely stimulated SMO/Hedgehog pathway that was responsible for the proliferation and metastasis of OS.

RAB23 coordinates early osteogenesis by repressing FGF10-pERK1/2 and GLI1.

Mutations in the gene encoding Ras-associated binding protein 23 (RAB23) cause Carpenter Syndrome, which is characterized by multiple developmental abnormalities including polysyndactyly and defects in skull morphogenesis. To understand how RAB23 regulates skull development, we generated Rab23-deficient mice that survive to an age where skeletal development can be studied. Along with polysyndactyly, these mice exhibit premature fusion of multiple sutures resultant from aberrant osteoprogenitor proliferation and elevated osteogenesis in the suture. FGF10-driven FGFR1 signaling is elevated in Rab23-/-sutures with a consequent imbalance in MAPK, Hedgehog signaling and RUNX2 expression. Inhibition of elevated pERK1/2 signaling results in the normalization of osteoprogenitor proliferation with a concomitant reduction of osteogenic gene expression, and prevention of craniosynostosis. Our results suggest a novel role for RAB23 as an upstream negative regulator of both FGFR and canonical Hh-GLI1 signaling, and additionally in the non-canonical regulation of GLI1 through pERK1/2.

In many animals, the skull is made of several separate bones that are loosely joined during childhood and only fuse into one piece when the animal stops growing. A genetic disease called Carpenter syndrome causes the bones of the skull to fuse early in life, stopping it from growing correctly. Carpenter syndrome is often caused by changes to the gene responsible for making a protein called RAB23. RAB23 helps move other molecules and cell components between different parts of the cell, and is therefore involved in a number of cellular processes. Previous studies suggest that RAB23 has a role in many parts of the body during development. Yet, it is unclear which cells in the skull depend on RAB23 activity and how this protein is controlled. To answer this question, Hasan et al. grew pieces of developing skull bones that had been taken from mice lacking the RAB23 protein in the laboratory. Examining these samples revealed that RAB23 is active in cells called osteoblasts that add new bone to the edge of each piece of the skull as it grows. Hasan et al. also found that RAB23 regulates two cellular signaling pathways ­ called the hedgehog pathway and the fibroblast growth factor pathway ­ that interact with one another and co-ordinate skull development. These findings show how RAB23 controls the growth and fusion of skull bones in developing animals. This could improve our understanding of the role RAB23 plays in other processes during development. It also sheds light on the mechanisms of Carpenter syndrome which may inform new approaches for treating patients.

Gene of the month: GLI-1.

The Glioma-associated homologue-1 (GLI-1) gene was first discovered to be amplified in glioblastoma multiforme. It encodes for a zinc-finger transcription factor in the Kruppel family of proteins and is important in the sonic hedgehog signalling pathway. GLI-1 also plays a role in several other pathways and is important for proliferation, migration, invasion, growth and angioinvasion, and cancer stem cell self-renewal in a variety of malignancies. GLI-1 is amplified in several malignancies, including an epithelioid, pericytomatous soft tissue neoplasm that can exhibit malignant behaviour. More recently, GLI-1 fusions with other partner genes have been found in three rare tumours: a pericytomatous tumour with a t(7;12) translocation, where it partners with Actin beta 1, and gastroblastoma and plexiform fibromyxoma, where the partner gene is metastasis-associated lung adenocarcinoma transcript 1, respectively.

Gli promotes tumor progression through regulating epithelial-mesenchymal transition in non-small-cell lung cancer.

INTRODUCTION: Lung cancer is the leading causes of cancer-related deaths globally. The most frequent histologic type of lung cancer is non-small-cell lung cancer (NSCLC). NSCLC often undergo epithelial-mesenchymal transition (EMT). The components that control this process are thus promising therapeutic targets. MATERIALS AND METHODS: Gli/EMT protein expression levels were examined by western blot in paired NSCLC patient tissues and NSCLC cell lines. Functional analyses were performed to investigate SHH/Gli signaling and EMT in NSCLC cell lines. MTS cell viability, luciferase reporter, and western blot assays were performed to analyze pathway activity, while wound healing and transwell assays were executed to measure cell migration and invasion. RESULTS: Higher Gli1 expressions were detected in tumor samples than in paired normal tissues. Differential expression of EMT biomarkers and activation of p-AKT were observed in tumor tissues. N-Shh stimulation of cells significantly increased reporter activity in NSCLC cell lines, while Gli-i treatment of transfected cells showed less relative reporter activity. When subjected to both Gli-i and N-Shh treatment, NSCLC cell lines continued to demonstrate decreased Gli transcriptional activity. Gli inhibition is associated with decreased expression level of p-AKT, N-cadherin and Vimentin. Knockdown of both Gli1 and Gli2 showed decreased EMT, migrative and invasive ability. Cells stimulated by N-Shh demonstrated greater mobility. In addition, AKT-i treated cells also demonstrated inhibited EMT activity. CONCLUSIONS: This study provides evidence for aberrant upregulation of the Gli signaling pathway and a strong association between expression of Gli versus AKT and EMT markers in NSCLC.

Cell-Autonomous Hedgehog Signaling Is Not Required for Cyst Formation in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: PKD1 or PKD2, the two main causal genes for autosomal dominant polycystic kidney disease (ADPKD), encode the multipass transmembrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Polycystins localize to the primary cilium, an organelle essential for cell signaling, including signal transduction of the Hedgehog pathway. Mutations in ciliary genes that build and maintain the cilium also cause renal cystic disease through unknown pathways. Although recent studies have found alterations in Hedgehog signaling in ADPKD-related models and tissues, the relationship between Hedgehog and polycystic kidney disease is not known. METHODS: To examine the potential role of cell-autonomous Hedgehog signaling in regulating kidney cyst formation in vivo in both early- and adult-onset mouse models of ADPKD, we used conditional inactivation of Pkd1 combined with conditional modulation of Hedgehog signaling components in renal epithelial cells, where mutations in Pkd1 initiate cyst formation. After increasing or decreasing levels of Hedgehog signaling in cells that underwent inactivation of Pkd1, we evaluated the effects of these genetic manipulations on quantitative parameters of polycystic kidney disease severity. RESULTS: We found that in Pkd1 conditional mutant mouse kidneys, neither downregulation nor activation of the Hedgehog pathway in epithelial cells along the nephron significantly influenced the severity of the polycystic kidney phenotype in mouse models of developmental or adult-onset of ADPKD. CONCLUSIONS: These data suggest that loss of Pkd1 function results in kidney cysts through pathways that are not affected by the activity of the Hedgehog pathway.

MTA1 promotes the invasion and migration of oral squamous carcinoma by inducing epithelial-mesenchymal transition via the hedgehog signaling pathway.

The metastasis-associated gene 1 (MTA1) has previously been recognized as an oncogene in many tumors, and aberrant MTA1 expression has been related to invasion and migration; however, its role and underlying molecular mechanism in oral squamous carcinoma (OSCC) remain largely unexplored. In this work, we determined the expression of MTA1 in OSCC tissues and cell lines. The effect of MTA1 on metastasis and the role of MTA1 in the epithelial-to-mesenchymal transition (EMT) of OSCC cells were evaluated by assays both in vitro and in vivo. We also identified the key Hedgehog signaling pathway-related protein involved in the MTA1-induced EMT. We found that MTA1 expression was upregulated and positively related to the metastasis in OSCC tissues and cell lines. MTA1 overexpression promoted OSCC invasion, migration, and induced EMT, while its silencing had the opposite effect both in vitro and in vivo. Additionally, our data further revealed the relevant molecular mechanism, Hedgehog(Hh) signaling pathway contributed to the effect of MTA1 on the aggressive phenotypes of OSCC cells.These findings indicate that MTA1 enhances OSCC cells invasion and migration by inducing EMT via the Hedgehog signaling pathway, which suggests MTA1 may be an effective anti-OSCC therapeutic target.

Antidepressant and Neuroprotective Effects of Naringenin via Sonic Hedgehog-GLI1 Cell Signaling Pathway in a Rat Model of Chronic Unpredictable Mild Stress.

Depression is one of the most prevalent and crucial public health problem connected to significant mortality and co-morbidity. Recently, numerous studies suggested that dietary flavanones exhibit neuroprotective and antidepressant effects against various psycho-physiological conditions including depression. The present study is focused on the antidepressant and neuroprotective effects of naringenin (NAR) and the involvement of sonic hedgehog (Shh) signaling in the chronic unpredictable mild stress (CUMS)-induced depression. Twenty-four male Wistar rats were randomly assigned into four groups: CON group (saline s.c.), NAR group (NAR 50 mg/kg, p.o.), CUMS group (subjected to CUMS along with saline p.o.), and CUMS + NAR group (NAR 50 mg/kg p.o. along with CUMS) for 28 days including 1-week pre-treatment with NAR. The results showed that NAR was found to inhibit behavioral abnormalities including increased despair in force swim test, and reduced locomotor activity caused by CUMS in open field test. Moreover, Morris water maze revealed that NAR also mitigates CUMS-associated cognitive impairment. In addition to the antidepressant-like effect, NAR mitigates morphological anomalies in the hippocampal CA1 region and cortex. Furthermore, we observed brain-derived neurotrophic factor (BDNF), Shh, GLI1, NKX2.2, and PAX6 were downregulated in the hippocampus of CUMS-exposed rats, which can be upregulated by NAR pre-treatment. GLI1 is main downstream signaling component of Shh signaling cascade, which further regulates the expression of homeodomain transcription factors PAX6 and NKX2.2.

The Hedgehog target Gli1 is not required for bleomycin-induced lung fibrosis.

Sonic Hedgehog (SHH) signaling, a developmental pathway promoting lung mesenchymal expansion and differentiation during embryogenesis, has been increasingly recognized as a profibrotic factor in mature lung, where it might contribute to the pathogenesis of lung fibrosis. Pathway inhibition at the level of the downstream Gli transcription factors Gli1 and Gli2 (by GANT61) ameliorates lung fibrosis in the bleomycin model, whereas inhibition proximally at the level of HH ligand (by anti Hh antibody 5E1) or Smo (by GDC-0449) of the canonical pathway does not, implicating Gli1 and/or Gli2 as a key target. The fact that both the Gli1-labelled cell lineage and Gli1 expressing cells expand during fibrosis formation and contribute significantly to the pool of myofibroblasts in the fibrosis scars suggests a fibrogenic role for Gli1. Therefore to further dissect the roles of Gli1 and Gli2 in lung fibrosis we evaluated Gli1 KO and control mice in the bleomycin model. Monitoring of Gli1+/+ (n = 12), Gli1lZ/+ (n = 37) and Gli1lZ/lZ (n = 18) mice did not reveal differences in weight loss or survival. Lung evaluation at the 21-day endpoint did not show differences in lung fibrosis formation (as judged by morphology and trichrome staining), Ashcroft score, lung collagen content, lung weight, BAL protein content or BAL cell differential count. Our data suggest that Gli1 is not required for bleomycin-induced lung fibrosis.

Tick-tock hedgehog-mutual crosstalk with liver circadian clock promotes liver steatosis.

BACKGROUND & AIMS: The mammalian circadian clock controls various aspects of liver metabolism and integrates nutritional signals. Recently, we described Hedgehog (Hh) signaling as a novel regulator of liver lipid metabolism. Herein, we investigated crosstalk between hepatic Hh signaling and circadian rhythm. METHODS: Diurnal rhythms of Hh signaling were investigated in liver and hepatocytes from mice with ablation of Smoothened (SAC-KO) and crossbreeds with PER2::LUC reporter mice. By using genome-wide screening, qPCR, immunostaining, ELISA and RNAi experiments in vitro we identified relevant transcriptional regulatory steps. Shotgun lipidomics and metabolic cages were used for analysis of metabolic alterations and behavior. RESULTS: Hh signaling showed diurnal oscillations in liver and hepatocytes in vitro. Correspondingly, the level of Indian Hh, oscillated in serum. Depletion of the clock gene Bmal1 in hepatocytes resulted in significant alterations in the expression of Hh genes. Conversely, SAC-KO mice showed altered expression of clock genes, confirmed by RNAi against Gli1 and Gli3. Genome-wide screening revealed that SAC-KO hepatocytes showed time-dependent alterations in various genes, particularly those associated with lipid metabolism. The clock/hedgehog module further plays a role in rhythmicity of steatosis, and in the response of the liver to a high-fat diet or to differently timed starvation. CONCLUSIONS: For the first time, Hh signaling in hepatocytes was found to be time-of-day dependent and to feed back on the circadian clock. Our findings suggest an integrative role of Hh signaling, mediated mainly by GLI factors, in maintaining homeostasis of hepatic lipid metabolism by balancing the circadian clock. LAY SUMMARY: The results of our investigation show for the first time that the Hh signaling in hepatocytes is time-of-day dependent, leading to differences not only in transcript levels but also in the amount of Hh ligands in peripheral blood. Conversely, Hh signaling is able to feed back to the circadian clock.

LAP2 Proteins Chaperone GLI1 Movement between the Lamina and Chromatin to Regulate Transcription.

Understanding transcription factor navigation through the nucleus remains critical for developing targeted therapeutics. The GLI1 transcription factor must maintain maximal Hedgehog pathway output in basal cell carcinomas (BCCs), and we have previously shown that resistant BCCs increase GLI1 deacetylation through atypical protein kinase Cι/λ (aPKC) and HDAC1. Here we identify a lamina-associated polypeptide 2 (LAP2) isoform-dependent nuclear chaperoning system that regulates GLI1 movement between the nuclear lamina and nucleoplasm to achieve maximal activation. LAP2ß forms a two-site interaction with the GLI1 zinc-finger domain and acetylation site, stabilizing an acetylation-dependent reserve on the inner nuclear membrane (INM). By contrast, the nucleoplasmic LAP2α competes with LAP2ß for GLI1 while scaffolding HDAC1 to deacetylate the secondary binding site. aPKC functions to promote GLI1 association with LAP2α, promoting egress off the INM. GLI1 intranuclear trafficking by LAP2 isoforms represents a powerful signal amplifier in BCCs with implications for zinc finger-based signal transduction and therapeutics.

Gli1 promotes colorectal cancer metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling.

Colorectal cancer(CRC) is one of the most commonly diagnosed cancers in human beings and metastasis is the main death reason. Recently, Gli1 has been reported to be a key regulator of various cancer biologies and genes expressions. However, the detailed molecular mechanism of Gli1 in CRC metastasis remains largely unknown. In this study, we aimed to investigate the role of Gli1 in CRC metastasis. We used qRT-PCR, Immunohistochemistry and Western blot to test the expression levels of Gli1, Foxm1 and other target genes in the tissues and cells; Lentivirus stable transfection to change the expression levels of Gli1 and Foxm1; Wound-healing, cell invasion, migration assays and tail vein metastatic assay to test the role of Gli1 in CRC metastasis in vitro and vivo. We demonstrated that Gli1 was significantly overexpressed in colorectal cancer tissues and cells. Foxm1 level had a positive correlation with Gli1. Furthermore, we found that Gli1 promotes colorectal cancer cells metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling. Thus, we proved that Gli1 plays important role in CRC metastasis and provided a new visual field on the therapy of CRC metastasis.

Blockade of the Hedgehog pathway downregulates estrogen receptor alpha signaling in breast cancer cells.

Anti-estrogen treatment, exemplified by tamoxifen, is a well-established adjuvant therapy for estrogen receptor alpha (ERα)-positive breast cancer. However, the effectiveness of this drug is limited due to the development of resistance. The Hedgehog (HH) signaling pathway is critical in embryonic development, and aberrant activation of this transduction cascade is linked to various malignancies. However, it remains unclear whether HH signaling is activated in human breast cancer and related to tamoxifen resistance. Deciphering how this pathway may be involved in breast cancer is a crucial step towards the establishment of targeted combinatorial treatments for this disease. Here, we show that the expression of the HH signaling effector protein GLI1 is higher in tamoxifen resistant compared to sensitive cells. Tamoxifen resistant cells have stronger ERα transcriptional activity relative to sensitive cells, even though the ERα expression is similar in both cell types. Knockdown of GLI1 attenuates cell proliferation and reduces ERα transcriptional activity in both sensitive and resistant cells, irrespective of estrogen stimulation. Combinatorial treatment of tamoxifen and the GLI antagonist GANT61 further suppresses the growth of sensitive and resistant cells relative to administration of only tamoxifen, and this was irrespective of estrogen stimulation. Moreover, a positive correlation between GLI1 and ERα expression was identified in breast cancer samples. Additionally, high GLI1 expression predicted worse distant metastasis-free survival in breast cancer patients. These data suggest that the HH pathway may be a new candidate for therapeutic targeting and prognosis in ERα-positive breast cancer.