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Epithelial‑mesenchymal transition (EMT) provides a valuable source of fibroblasts that produce extracellular matrix in airway walls. The Sonic hedgehog (SHH) signaling pathway plays an essential role in regulating tissue turnover and homeostasis. SHH is strikingly upregulated in the bronchial epithelia during asthma. Snail1 is a major target of SHH signaling, which regulates EMT and fibroblast motility. The present study was designed to ascertain whether the combination of house dust mite (HDM) and transforming growth factor β1 (TGF‑β1) could induce EMT via the SHH signaling pathway in human bronchial epithelial cells (HBECs). HBEC cultures were treated with HDM/TGF‑β1 for different periods of time. The involvement of SHH signaling and EMT biomarkers was evaluated by quantitative real‑time PCR, western blotting and immunofluorescence staining. Small‑interfering RNA (siRNA) for glioma‑associated antigen‑1 (Gli1) or cyclopamine was used to inhibit SHH signaling in HBECs. HBECs stimulated by HDM/TGF‑β1 exhibited morphological features of EMT. E‑cadherin (an epithelial marker) was decreased after a 72‑h exposure to HDM/TGF‑β1 compared to that in the control cells, and the expression of type I collagen and FSP1 (mesenchymal markers) was increased. HDM/TGF‑β1 activated the SHH signaling pathway in HBECs, which led to Gli1 nuclear translocation and the transcriptional activation of Snail1 expression. Moreover, gene silencing or the pharmacological inhibition of Gli1 ameliorated EMT. In summary, these findings suggest that HDM/TGF‑β1 may induce EMT in HBECs via an SHH signaling mechanism. Inhibition of SHH signaling may be a novel therapeutic method for preventing airway remodeling in asthma.

Purmorphamine (PUR), an agonist of the Smoothened (Smo) receptor, has been shown to function as a neuroprotectant in acute experimental ischemic stroke. Its role in hypoxic-ischemic (HI) brain injury in neonatal mice remains unknown. Here we show that PUR attenuated acute brain injury, with a decrease in Bax/Bcl-2 ratio as well as inhibition of caspase-3 activation. These beneficial effects of PUR were associated with suppressing neuro-inflammation and oxidative stress. PUR exerted long-term protective effects upon tissue loss and improved neurobehavioral outcomes as determined at 14 and 28 days post-HI insult. Moreover, PUR increased synaptophysin (Syn) and postsynaptic density (PSD) protein 95 expression in HI-treated mice and attenuated synaptic loss. PUR upregulated the expression of Shh pathway mediators, while suppression of the Shh signaling pathway with cyclopamine (Cyc) reversed these beneficial effects of PUR on HI insult. Our study suggests a therapeutic potential for short-term PUR administration in HI-induced injury as a result of its capacity to exert multiple protective actions upon acute brain injury, long-term memory deficits, and impaired synapses. Moreover, we provide evidence indicating that one of the mechanisms underlying these beneficial effects of PUR involves activation of the Shh signaling pathway.

Background/Aims: Neurite outgrowth and synaptogenesis are critical steps for functional recovery after stroke. Resveratrol promotes neurite outgrowth and synaptogenesis, but the underlying mechanism is not well understood, although the Sonic hedgehog (Shh) signaling pathway may be involved. Given that resveratrol activates sirtuin (Sirt)1, the present study examined whether this is mediated by Shh signaling. Methods: Primary cortical neuron cultures were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability and apoptosis were evaluated with Cell Counting Kit 8 and by terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Neurite outgrowth and synaptogenesis were assessed by immunocytochemistry and western blotting, which was also used to examine the expression of Sirt1 and Shh signaling proteins. Results: Resveratrol and the Smoothened (Smo) agonist purmophamine, which activates Shh signaling, increased viability, reduced apoptosis, and stimulated neurite outgrowth after OGD/R injury. Moreover, the expression of growth-associated protein(GAP)-43, synaptophysin, Shh, Patched (Ptc)-1, Smo, glioma-associated oncogene homolog (Gli)-1, and Sirt1 were upregulated under these conditions. These effects were reversed by treatment with the Smo inhibitor cyclopamine, whereas the Sirt1 inhibitor sirtinol reduced the levels of Shh, Ptc-1, Smo, and Gli-1. Conclusions: Resveratrol reduces neuronal injury following OGD/R injury and enhances neurite outgrowth and synaptogenesis by activating Shh signaling, which in turn induces Sirt1.

Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic stellate cells (PSCs). Gremlin 1 is widely overexpressed in cancer-associated stromal cells, including activated PSCs. In embryonic development, SHH is a potent regulator of Gremlin 1 through an interaction network. This subtle mechanism in the cancer microenvironment remains to be fully elucidated. The present study investigated the association between Gremlin 1 and SHH, and the effect of Gremlin 1 in pancreatic cancer. The expression of Gremlin 1 in different specimens was measured using immunohistochemistry. The correlations among clinico-pathological features and levels of Gremlin 1 were evaluated. Primary human PSCs and pancreatic cancer cell lines were exposed to SHH, cyclopamine, GLI family zinc finger-1 (Gli-1) small interfering RNA (siRNA), and Gremlin 1 siRNA to examine their associations and effects using an MTT assay, reverse transcription-quantitative polymerase chain reaction analysis, western blot analysis, and migration or invasion assays. The results revealed the overexpression of Gremlin 1 in pancreatic cancer tissues, mainly in the stroma. The levels of Gremlin 1 were significantly correlated with survival rate and pT status. In addition, following activation of the PSCs, the expression levels of Gremlin 1 increased substantially. SHH acts as a potent promoter of the expression of Gremlin 1, and cyclopamine and Gli-1 siRNA modulated this effect. In a screen of pancreatic cancer cell lines, AsPC-1 and BxPC-3 cells expressed high levels of Gremlin 1, but only AsPC-1 cells exhibited a high expression level of SHH. The results of the indirect co-culture experiment suggested that paracrine SHH from the AsPC-1 cells induced the expression of Gremlin 1 in the PSCs. Furthermore, Gremlin 1 siRNA negatively regulated the proliferation and migration of PSCs, and the proliferation, invasion and epithelial-mesenchymal transition of AsPC-1 and BxPC-3 cells. Based on the data from the present study, it was concluded that an abnormal expression level of Gremlin 1 in pancreatic cancer was induced by SHH signaling, and that the overexpression of Gremlin 1 enabled pancreatic cancer progression.

Background/Aims: Injuries of the brain and spinal cord result in the formation of glial (reactive gliosis) and fibrotic (formed by fibroblasts) scars. Recent studies have shown that the fibrotic scar was much more important for hindering regeneration after brain or spinal cord injury than the astrocytic scar. However, it has been given much less attention for effects and mechanism of fibroblasts during formation of the fibrotic scar. Resveratrol may be a potential anti-scarring agent in burn-related scarring and keloid fibroblasts. However, it is unclear whether and how resveratrol affects formation of the fibrotic scar after brain or spinal cord injury. Earlier studies have shown that the activated Shh signaling has anti-apoptosis, anti-oxidation, anti-inflammation properties. Moreover, resveratrol can activate the Shh signaling. However, it is unclear how resveratrol activates the Shh signaling. Resveratrol is a activator of Sirt1. It is unknown whether resveratrol activates the Shh signaling via Sirt1. Methods: NIH3T3 cells, a fibroblast cell line, were used as model cells and treated with drugs. Cell viability was assessed by Cell Counting Kit 8. The expressions and activity of Shh signaling pathway proteins were evaluated by immunocytochemistry and Western blotting. Transcriptional activity of Gli-1 was detected with Dual-Luciferase Reporter Gene Assay Kit. Results: Resveratrol, Sirt1 agonist STR1720 and recombinant mouse Shh protein, an activator of hedgehog signaling, enhanced the viability of NIH3T3 cells, promoted Smo to translocated to the primary cilia and Gli-1 entered into the nuclei from cytoplasm, and upregulated expressions of Shh, Ptc-1, Smo, and Gli-1 proteins, which can be reversed by Smo antagonist cyclopamine and Sirt1 antagonist Sirtinol. Additionally, resveratrol increased transcriptional activity of Gli-1. Conclusion: We indicate in the first time that it may be mediated by Sirt1 for resveratrol activating the Shh signaling to enhance viability of NIH3T3 cells, and Sirt1 may be a regulator for upstream of the Shh signaling pathway.This study provides a basis for further investigating effects and mechanism of resveratrol during the formation of fibrous scar after brain or spinal cord injury.

Background/Aims: There is interest in drugs and rehabilitation methods to enhance neurogenesis and improve neurological function after brain injury or degeneration. Resveratrol may enhance hippocampal neurogenesis and improve hippocampal atrophy in chronic fatigue mice and prenatally stressed rats. However, its effect and mechanism of neurogenesis after stroke is less well understood. Sonic hedgehog (Shh) signaling is crucial for neurogenesis in the embryonic and adult brain, but relatively little is known about the role of Shh signaling in resveratrol-enhanced neurogenesis after stroke. Methods: Neural stem cells (NSCs) before oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro were pretreated with resveratrol with or without cyclopamine. Survival and proliferation of NSCs was assessed by the CCK8 assay and BrdU immunocytochemical staining. The expressions and activity of signaling proteins and mRNAs were detected by immunocytochemistry, Western blotting, and RT-PCR analysis. Results: Resveratrol significantly increased NSCs survival and proliferation in a concentration-dependent manner after OGD/R injury in vitro. At the same time, the expression of Patched-1, Smoothened (Smo), and Gli-1 proteins and mRNAs was upregulated, and Gli-1 entered the nucleus, which was inhibited by cyclopamine, a Smo inhibitor. Conclusion: Shh signaling mediates resveratrol to increase NSCs proliferation after OGD/R injury in vitro.

Sonic hedgehog (SHH) signaling pathway is crucial to growth and patterning during organogenesis. Aberrant activation of the SHH signaling pathway can result in tumor formation. We examined the expression of SHH signaling molecules and investigated the involvement of the SHH pathway in the proliferation of ameloblastoma, the most common benign tumor of the jaws. We used immunohistochemistry on ameloblastoma specimens and immunocytochemistry and reverse transcription-PCR on the ameloblastoma cell line AM-1. We also used the inhibitors of SHH signaling, SHH neutralizing antibody and cyclopamine, to assess the effects of SHH on the proliferation of AM-1 cells. We detected expression of SHH, patched, GLI1, GLI2 and GLI3 in the ameloblastoma specimens and AM-1 cells. The proliferation of these cells was significantly inhibited in the presence of SHH neutralizing antibody or cyclopamine; this was confirmed by BrdU incorporation assays. Furthermore, in the presence of SHH neutralizing antibody, nuclear translocation of GLI1 and GLI2 was abolished, apoptosis was induced, BCL-2 expression decreased and BAX expression increased. Our results suggest that the SHH signaling pathway is constitutively active in ameloblastoma and plays an anti-apoptotic role in the proliferation of ameloblastoma cells through autocrine loop stimulation.

Fungiform papillae are epithelial specializations that develop in a linear pattern on the anterior mammalian tongue and differentiate to eventually contain taste buds. Little is known about morphogenetic and pattern regulation of these crucial taste organs. We used embryonic rat tongue, organ cultures to test roles for bone morphogenetic proteins, BMP2, 4 and 7, and antagonists noggin and follistatin, in development of papillae from a stage before morphological initiation (E13) or from a stage after the pre-papilla placodes have formed (E14). BMPs and noggin proteins become progressively restricted to papilla locations during tongue development. In E13 cultures, exogenous BMPs or noggin induce increased numbers of fungiform papillae, in a concentration-dependent manner, compared to standard tongue cultures; BMPs, but not noggin, lead to a decreased tongue size at this stage. In E14 cultures, however, exogenous BMP2, 4 or 7 each inhibits papilla formation so that there is a decrease in papilla number. Noggin substantially increases number of papillae in E14 cultures. Using beads for a highly localized protein delivery, papillae are inhibited in the surround of BMP-soaked beads and induced in large clusters around noggin-soaked beads. Follistatin, presented in culture medium or by bead, does not alter papilla formation or number. In all fungiform papillae that form under various culture conditions, the molecular marker, sonic hedgehog, is within each papilla. However, the BMP inhibitory effect on papillae is not prevented by disrupting sonic hedgehog signaling through addition of cyclopamine to cultures. BMPs and noggin alter cell proliferation in tongue epithelium in opposite ways, demonstrated with Ki67 immunostaining. We propose that the BMPs and noggin, colocalized within papilla placodes and the fungiform papillae per se, have opposing inhibitory and activating or inducing roles in papilla development in linear patterns. We present a model for these effects.