Sonic Hedgehog Inhibitor Cyclopamine Research Articles

Sonic hedgehog signaling promotes angiogenesis of endothelial progenitor cells to improve pressure ulcers healing by PI3K/AKT/eNOS signaling.

Pressure ulcer is a severe disease in the paralyzed and aging populations. Endothelial progenitor cells (EPCs) are able to regulate ulcer healing by modulating angiogenesis, but the molecular mechanism is still obscure. Sonic hedgehog (SHH) signaling contributes to angiogenesis in various diseases and has been identified to modulate EPCs function. Here, we aimed to explore the significance of SHH signaling in EPCs function during pressure ulcers. The EPCs were isolated and characterized by the expression of DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1. Cell proliferation was detected by cell counting kit 8 (CCK-8). The DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 were analyzed by immunofluorescent analysis. The angiogenesis of EPCs was analyzed by tube formation assay. The pressure ulcers rat model was constructed, the wound injury was analyzed by H&E staining and angiogenesis was analyzed by the accumulation of CD31 based on immunofluorescent analysis. The expression of patched-1 and Gli-1 was enhanced by SHH activator SAG but reduced by SHH inhibitor cyclopamine in the EPCsThe PI3K, Akt, eNOS expression and the Akt phosphorylation were induced by SAG, while the treatment of cyclopamine presented a reversed result. The proliferation and migration of EPCs were enhanced by SAG but repressed by cyclopamine or PI3K/AKT/eNOS signaling inhibitor Y294002, in which the co-treatment of Y294002 could reverse the effect of SAG. Thus, we found that SHH signaling activated angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling. SHH signaling may serve as the potential target for attenuating pressure ulcers.

Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

Active intestinal stem cells are prone to injury by ionizing radiation. We previously showed that upon radiation-induced injury, normally quiescent reserve intestinal stem cells (rISCs) (marked by BMI1) are activated by Musashi-1 (MSI1) and exit from the quiescent state to regenerate the intestinal epithelium. This study aims to further establish the mechanism that regulates activation of Bmi1-CreER;Rosa26eYFP (Bmi1-CreER) rISCs following γ radiation-induced injury. Bmi1-CreER mice were treated with tamoxifen to initiate lineage tracing of BMI1 (eYFP+) cells and exposed to 12 Gy of total body γ irradiation or sham. Intestinal tissues were collected and analyzed by immunofluorescence, Western blot, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and chromatin immunoprecipitation real-time polymerase chain reaction. After irradiation, increased expression of Msi1 in eYFP+ cells was accompanied by increased expression of Axin2, a WNT marker. Promoter studies of the Msi1 gene indicated that Msi1 is a WNT target gene. Coculture of stromal cells isolated from irradiated mice stimulated Bmi1-CreER-derived organoid regeneration more effectively than those from sham mice. Expression of WNT ligands, including Wnt2b, Wnt4, Wnt5a, and Rspo3, was increased in irradiated stromal cells compared with sham-treated stromal cells. Moreover, expression of the Sonic hedgehog (SHH) effector Gli1 was increased in stromal cells from irradiated mice. This was correlated with an increased expression of SHH in epithelial cells postirradiation, indicating epithelial-stromal interaction. Finally, preinjury treatment with SHH inhibitor cyclopamine significantly reduced intestinal epithelial regeneration and Msi1 expression postirradiation. Upon ionizing radiation-induced injury, intestinal epithelial cells increase SHH secretion, stimulating stromal cells to secrete WNT ligands. WNT activators induce Msi1 expression in the Bmi1-CreER cells. This stromal-epithelial interaction leads to Bmi1-CreER rISCs induction and epithelial regeneration.

Abstract 1280: The intersection of the PI3K/mTOR and HIPPO pathways: a potential therapeutic target for medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor; it is genetically defined into 4 subgroups which defines the clinical course of the disease. Despite current treatment modalities such as surgical resection, radiation, and chemotherapy, there are still significant morbidities and adverse side-effects. Recently defined genetic pathways could possibly offer a better management and treatment of patients with MB. The HIPPO signaling pathway has recently been shown to intersect with PI3K and Sonic Hedgehog (SHH) pathways to modulate mechanistic target of rapamycin (mTOR), an important regulator in cell growth and migration. The aim of this study is to elucidate the interaction of Hippo and Akt/mTOR signaling pathways with SHH pathways in MB, and define the potential downstream targets of therapy by using combined inhibitors. In order to achieve our goals, we evaluated the expression of activated AKT/mTOR in MB tumors using immunohistochemistry (IHC), along with the expression of transcriptional co-activators yes-associated protein (YAP), a down-stream effector of the HIPPO pathway. Scratch and chemotactic migration were used to assess the motility of MB cells following treatment with inhibitors of the aforementioned pathways. Cell cycle analysis and proliferation were measured using EdU and MTT techniques, respectively. YAP-SiRNA treatment was used to inhibit HIPPO pathway. MicroRNA-29, which mediates the mTOR pathway by modulating PTEN levels, was investigated. Results demonstrated: 1. A significant number of MBs expressed activated pAKTSer473 and pmTORSer2448, along with YAP, the downstream effector of the HIPPO pathway; 2. Treatment with inhibitors of PI3K/AKT (LY294002) and mTOR (rapamycin), given with EGF, significantly reduced cellular motility; 3. SHH inhibitor cyclopamine given with LY294002 or rapamycin significantly reduced cellular motility; 4. Cell proliferation was suppressed by inhibition of the HIPPO pathway using 3 unique 27mer duplexes of YAP1 siRNA treatments; 5. Combined treatment of cyclopamine with either LY294002 or rapamycin caused maximum suppression of cellular growth. In conclusion, these results demonstrate that the Hippo and PI3K/Akt/mTOR pathways are over-expressed in MB, and that the combined targeting of mTOR/HIPPO pathway with SHH inhibitor may provide alternative strategies for the treatment of MB, and offers a targeted therapy for improved prognosis. Citation Format: Jennifer S. Ronecker, Paul Lee, Sudeepta Sridhara, Michael LaBagnara, Raj Murali, Meena Jhanwar-Uniyal. The intersection of the PI3K/mTOR and HIPPO pathways: a potential therapeutic target for medulloblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1280.

Sonic Hedgehog Promotes Cementoblastic Differentiation via Activating the BMP Pathways.

Although sonic hedgehog (SHH), an essential molecule in embryogenesis and organogenesis, stimulates proliferation of human periodontal ligament (PDL) stem cells, the effects of recombinant human SHH (rh-SHH) on osteoblastic differentiation are unclear. To reveal the role of SHH in periodontal regeneration, expression of SHH in mouse periodontal tissues and its effects on the osteoblastic/cementoblastic differentiation in human cementoblasts were investigated. SHH is immunolocalized to differentiating cementoblasts, PDL cells, and osteoblasts of the developing mouse periodontium. Addition of rh-SHH increased cell growth, ALP activity, and mineralization nodule formation, and upregulated mRNA expression of osteoblastic and cementoblastic markers. The osteoblastic/cementoblastic differentiation of rh-SHH was abolished by the SHH inhibitor cyclopamine (Cy) and the BMP antagonist noggin. rh-SHH increased the expression of BMP-2 and -4 mRNA, as well as levels of phosphorylated Akt, ERK, p38, and JNK, and of MAPK and NF-κB activation, which were reversed by noggin, Cy, and BMP-2 siRNA. Collectively, this study is the first to demonstrate that SHH can promote cell growth and cell osteoblastic/cementoblastic differentiation via BMP pathway. Thus, SHH plays important roles in the development of periodontal tissue, and might represent a new therapeutic target for periodontitis and periodontal regeneration.

GALNT1-Mediated Glycosylation and Activation of Sonic Hedgehog Signaling Maintains the Self-Renewal and Tumor-Initiating Capacity of Bladder Cancer Stem Cells.

The existence of bladder cancer stem cells (BCSC) has been suggested to underlie bladder tumor initiation and recurrence. Sonic Hedgehog (SHH) signaling has been implicated in promoting cancer stem cell (CSC) self-renewal and is activated in bladder cancer, but its impact on BCSC maintenance is unclear. In this study, we generated a mAb (BCMab1) against CD44(+) human bladder cancer cells that recognizes aberrantly glycosylated integrin α3β1. The combination of BCMab1 with an anti-CD44 antibody identified a BCMab1(+)CD44(+) cell subpopulation as BCSCs with stem cell-like properties. Gene expression analysis revealed that the hedgehog pathway was activated in the BCMab1(+)CD44(+) subpopulation and was required for BCSC self-renewal. Furthermore, the glycotransferase GALNT1 was highly expressed in BCMab1(+)CD44(+) cells and correlated with clinicopathologic features of bladder cancers. Mechanistically, GALNT1 mediated O-linked glycosylation of SHH to promote its activation, which was essential for the self-renewal maintenance of BCSCs and bladder tumorigenesis. Finally, intravesical instillation of GALNT1 siRNA and the SHH inhibitor cyclopamine exerted potent antitumor activity against bladder tumor growth. Taken together, our findings identify a BCSC subpopulation in human bladder tumors that appears to be responsive to the inhibition of GALNT1 and SHH signaling, and thus highlight a potential strategy for preventing the rapid recurrence typical in patients with bladder cancer.

Sonic Hedgehog Signaling: Evidence for Its Protective Role in Endotoxin Induced Acute Lung Injury in Mouse Model.

ObjectiveTo investigate the protective role of the sonic hedgehog (SHH) signaling associated with a lipopolysaccharide (LPS)-induced acute lung injury (ALI) in a mouse model.MethodsMale BALB/c mice were randomly divided into four groups: control, LPS, LPS-cyclopamine group and cyclopamine group. ALI was induced by LPS ip injection (5 mg/kg). The sonic hedgehog inhibitor cyclopamine (50 mg/kg) was given to the LPS-cyclopamine group at 30 min after LPS injection as well as normal mice as control. Lung injury was observed histologically in hematoxylin and eosin (HE) stained tissue sections, semi-quantified by lung tissue injury score, and the lung tissue mass alteration was measured by wet to dry weight ratio (W/D). mRNA expression levels of TNF-α, SHH, Patched (PTC) and GLI1 in lung tissue were studied with real time quantitative PCR (RT-PCR), while the protein expression of SHH and GLI1 was determined by western blot analysis.ResultsLung tissue injury score, thickness of alveolar septa, W/D, and TNF-α mRNA expression levels were significantly higher in the ALI mice than the normal mice (P<0.05). The mRNA expression levels of SHH, PTC, and GLI1 in the ALI mice were significantly higher at 12h and 24h after LPS injection, but not at the 6h time point. Protein production of SHH and GLI1 at 6h, 12h, and 24h in the lungs of ALI mice significantly increased, in a time-dependent manner, compared with that in normal mice. Cyclopamine alone has no effect on pathological changes in normal mice. Intervention with cyclopamine in ALI mice led to a reduction in mRNA levels of SHH, PTC, and GLI1 as well as SHH and GLI1 protein levels; meanwhile, the pathological injury scores of lung tissues, thickness of alveolar septa, W/D, and mRNA expression levels of TNF-α increased compared with mice receiving LPS only.ConclusionThe SHH signaling pathway was activated in response to LPS-induced ALI, and up-regulation of SHH expression could alleviate lung injury and be involved in the repair of injured lung tissue.

Highly lymphatic metastatic pancreatic cancer cells possess stem cell-like properties

Cancer stem cells are thought to be the origin of tumor metastasis. However, evidence of cancer stem cells as the source of lymphatic metastasis in pancreatic cancer is not clear. In this study, we examined the stem cell-like properties of the highly lymphatic metastatic pancreatic cancer cells BxPC-3-LN. Compared with the parental BxPC-3 cells, the BxPC-3-LN cells showed stem cell-like properties, including high lymphatic metastasis potential, self-renewal ability and chemoresistance. In addition, the BxPC-3-LN cells also expressed higher levels of sonic hedgehog and migrating cancer stem cell surface markers (CD133 and CXCR4) compared to the parental BxPC-3 cells. The growth of BxPC-3-LN cells was significantly inhibited by gemcitabine combined with the sonic hedgehog inhibitor cyclopamine. The BxPC-3-LN cells expressed lower levels of let-7, miR-34, miR-107, miR-125, miR-128, miR-130, miR-132 and miR-141 than the parental BxPC-3 cells detected by microRNA PCR array, which were reported to have close relation to stem cell factors. This study provides evidence that cancer stem cells are the major sources of pancreatic cancer lymphatic metastasis, and microRNAs may regulate lymphatic metastasis in pancreatic cancer through modulating cancer stem cells.

Abstract 17495: Thrombospondin-1/CD36 Pathway Critically Contributes to SHH Signaling Defect and EPC Dysfunction on Wound Healing in Type 1 Diabetes

Introduction: Refractory wounds in diabetic patients is a significant clinical problem. We reported previously that sonic hedgehog (SHH), a morphogenic protein central to wound repair, is deficient in diabetes, but the underlying mechanisms are poorly understood. We hypothesized that thrombospondin-1 (TSP-1) through its receptor CD36 contributes to SHH signaling defect in cutaneous tissue and endothelial progenitor cells (EPCs), resulting in impaired wound healing in type 1 diabetic mice. Methods and Results: Cutaneous expression of SHH and its signaling molecules including Patched (Ptc), Smoothened (Smo), and Gli were significantly decreased in streptozotocin (STZ)-induced type 1 diabetic mice (male, C57BL/6J, 12 weeks) vs. their age-matched control littermates (Western blot analysis, n=5, p&lt;0.05 vs. control), which were reversed in STZ-treated TSP-1 -/- mice (homozygote, C57BL/6J background, n=5, p&lt;0.05 vs. STZ-treated WT mice). Excisional wound (6-mm punch biopsy) closure was significantly delayed in STZ mice (n=6, p&lt;0.05 vs. control) with reduced wound blood flow (Laser Doppler), which was not observed in STZ-treated TSP-1 -/- mice (n=5, p&lt;0.05 vs. STZ-treated WT mice). The protective effect of TSP-1 depletion on wound repair was abolished following topical treatment with the selective SHH inhibitor cyclopamine (5 μ M, 30 min/day, n=5, p&lt;0.05 vs. STZ-treated TSP-1 -/- mice). Isolated EPCs from bone marrow of STZ mice showed significantly impaired angiogenesis (Matrigel tube formation, n=4, p&lt;0.05 vs. control), which was rescued by exogenous SHH (1 mg/L, 24 hours, n=4, p&lt;0.05 vs. STZ-control). Increased EPC angiogenesis in STZ-treated TSP-1 -/- mice (n=5, p&lt;0.05 vs. STZ-WT) was inhibited by the selective SHH inhibitor cyclopamine (5 μ M, 24 hours, n=5, p&lt;0.05). SHH, Ptc, Smo and Gli levels were significantly increased in EPCs from TSP-1 -/- mice and CD36 -/- mice, but not CD47 -/- mice (n=6, p&lt;0.05 vs. WT). Normal EPCs treated with recombinant human TSP-1 protein (2.2 nM, 24 hour) exhibited impaired angiogenesis, an effect that was not observed in EPCs from CD36 -/- mice (n=5, p&lt;0.05). Conclusion: TSP-1/CD36 pathway critically contributes to SHH signaling defect, resulting in EPC dysfunction and impaired wound healing in type 1 diabetic mice.

Erythropoietin and sonic hedgehog mediate the neuroprotective effects of brain-derived neurotrophic factor against mitochondrial inhibition

Brain-derived neurotrophic factor (BDNF) deficiency and mitochondrial dysfunction have been implicated in the pathogenesis of Huntington's disease (HD). 3-Nitropropionic acid (3-NP) is a mitochondrial inhibitor commonly used as a pharmacological model mimicking HD. We have recently reported that preconditioning of primary rat cortical cultures with BDNF induces sonic hedgehog (SHH), which contributes to the protective effects of BDNF against 3-NP neurotoxicity. Because carbamylated erythropoietin (EPO) may induce SHH, we investigated whether BDNF-dependent SHH expression and 3-NP resistance require prior induction of EPO. We found that BDNF induced EPO expression at both mRNA and protein levels. BDNF-mediated SHH induction and 3-NP resistance were abolished by the soluble EPO receptor (sEPO-R), an EPO inhibitor. Recombinant rat EPO (rEPO) induced SHH and attenuated 3-NP neurotoxicity. The rEPO-dependent neuroprotection was suppressed by the SHH inhibitor cyclopamine (CPM); however, sEPO-R failed to affect SHH neuroprotection. Furthermore, the rEPO-dependent neuroprotection was not suppressed by the BDNF neutralizing antibody, which completely abolished BDNF-mediated 3-NP resistance at the same dosage. Overall, our results demonstrate that BDNF-dependent SHH expression and 3-NP resistance require prior induction of EPO, thus establishing a signaling cascade of “BDNF → EPO → SHH → 3-NP resistance” in rat cortical neurons.