Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. To directly test the effect of PTCH1 on accessible cholesterol, we measured the transport activity of PTCH1 using an imaging-based assay to follow the kinetics of cholesterol extraction from the plasma membrane of live cells by methyl-β-cyclodextrin. PTCH1 depletes accessible cholesterol in the outer leaflet of the membrane in a manner regulated by its ligand Sonic Hedgehog and the transmembrane potassium gradient. We propose that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export. Our results show that proteins can change accessible cholesterol levels in membranes to regulate signaling reaction.

Sterols, Oxysterols, and Accessible Cholesterol: Signalling for Homeostasis, in Immunity and During Development

In this article we discuss the concept of accessible plasma membrane cholesterol and its involvement as a signalling molecule. Changes in plasma membrane accessible cholesterol, although only being minor in the context of total cholesterol plasma membrane cholesterol and total cell cholesterol, are a key regulator of overall cellular cholesterol homeostasis by the SREBP pathway. Accessible cholesterol also provides the second messenger between patched 1 and smoothened in the hedgehog signalling pathway important during development, and its depletion may provide a mechanism of resistance to microbial pathogens including SARS-CoV-2. We revise the hypothesis that oxysterols are a signalling form of cholesterol, in this instance as a rapidly acting and paracrine version of accessible cholesterol.

A high level of KLF12 causes folic acid-resistant neural tube defects by activating the Shh signalling pathway in mice

Although adequate periconceptional folic acid (FA) supplementation has reduced the occurrence of pregnancies affected by neural tube defects (NTDs), the mechanisms underlying FA-resistant NTDs are poorly understood, and thus NTDs still remain a global public health concern. A high level of Krüppel-like factor 12 (KLF12) exerts deleterious effects on heath in most cases, but evidence for its roles in development has not been published. We observed KLF12-overexpressing mice showed disturbed neural tube development. KLF12-overexpressing foetuses died in utero at approximately 10.5 days post coitus, with 100% presenting cranial NTDs. Neither FA nor formate promoted normal neural tube closure in mutant foetuses. The RNA-seq results showed that a high level of KLF12 caused NTDs in mice via overactivating the sonic hedgehog (Shh) signalling pathway, leading to the upregulation of patched 1, GLI-Krüppel family member GLI1, hedgehog-interacting protein, etc., while FA metabolism-related enzymes did not express differently. PF-5274857, an antagonist of the Shh signalling pathway, significantly promoted dorsolateral hinge point formation and partially rescued the NTDs. The regulatory hierarchy between a high level of KLF12 and FA-resistant NTDs might provide new insights into the diagnosis and treatment of unexplained NTDs in the future.

CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids

The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis. Aberrant HH signaling can cause congenital malformations, such as digit anomalies and holoprosencephaly, and other diseases, including cancer. Signal transduction is initiated by HH ligand binding to the Patched 1 (PTCH1) receptor on primary cilia, thereby releasing inhibition of Smoothened (SMO), a HH pathway activator. Although cholesterol and several oxysterol lipids, which are enriched in the ciliary membrane, play a crucial role in HH activation, the molecular mechanisms governing the regulation of these lipid molecules remain unresolved. Here, we identify Canopy 4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that controls membrane sterol lipid levels. Cnpy4—/— embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway at the level of SMO in vitro, and elevates membrane levels of accessible sterol lipids such as cholesterol, an endogenous ligand involved in SMO activation. Thus, our data demonstrate that CNPY4 is a negative regulator that fine-tunes the initial steps of HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.

Novel PTCH1 mutations in Japanese familial nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, is inherited in an autosomal dominant manner and is characterized by a combination of developmental abnormalities and a predisposition to tumor formation. Hedgehog receptor Patched 1 (PTCH1) has been identified as the mutated gene in NBCCS. We identified the PTCH1_c.3298_3299insAAG_p.1099_1100insE mutation in the transmembrane region, which comprises a sterol transporter whose abnormal function is reportedly related to pathogenicity.

Full-length Dhh and N-terminal Shh act as competitive antagonists to regulate angiogenesis and vascular permeability

Aims The therapeutic potential of Hedgehog (Hh) signalling agonists for vascular diseases is of growing interest. However, molecular and cellular mechanisms underlying the role of the Hh signalling in vascular biology remain poorly understood. The purpose of the present article is to clarify some conflicting literature data. Methods and results With this goal, we have demonstrated that, unexpectedly, ectopically administered N-terminal Sonic Hh (N-Shh) and endogenous endothelial-derived Desert Hh (Dhh) induce opposite effects in endothelial cells (ECs). Notably, endothelial Dhh acts under its full-length soluble form (FL-Dhh) and activates Smoothened in ECs, while N-Shh inhibits it. At molecular level, N-Shh prevents FL-Dhh binding to Patched-1 (Ptch1) demonstrating that N-Shh acts as competitive antagonist to FL-Dhh. Besides, we found that even though FL-Hh ligands and N-Hh ligands all bind Ptch1, they induce distinct Ptch1 localization. Finally, we confirmed that in a pathophysiological setting, i.e. brain inflammation, astrocyte-derived N-Shh acts as a FL-Dhh antagonist. Conclusion The present study highlights for the first time that FL-Dhh and N-Hh ligands have antagonistic properties especially in ECs.

Icariin Promote Stem Cells Regeneration and Repair Acinar Cells in L-arginine / Radiation -Inducing Chronic Pancreatitis in Rats

Objective: Chronic Pancreatitis (CP) is a multifactorial disease. It was characterized by severe inflammation and acinar cell destruction. Thus, the present study was initiated to evaluating the ability of bone marrow-based mesenchymal stem cell (MSCs) combined with Icariin to restore and regenerate acinar cells in the pancreas of rats suffering chronic pancreatitis. Methods: Chronic pancreatitis was induced in rats via both L-arginine plus radiation, repeated L-arginine injection (2.5g/Kg body-weight, 1, 4,7,10,13,16,19 days), then, on day 21, rats were exposed to a single dose of gamma-radiation (6 Gy), which exacerbate injury of pancreatic acinar cells. One day after irradiation, rats were treated with either MSCs (1 × 107 /rat, once, tail vein injection) labeled PKH26 fluorescent linker dye and/or Icariin (100 mg/Kg, daily, orally) for 8 weeks. Results: Icariin promotes MSCs proliferation boosting its productivity in vitro. MSCs, and/or icariin treatments has regulated molecular factors TGF-β/PDGF and promoted the regeneration of pancreatic tissues by releasing PDX-1 and MafA involved in the recruitment of stem/progenitor cell in the tissue, and confirmed by histopathological examination. Moreover, a significant decrease in IL-8 and TNF-α cytokines with significant amelioration of myeloperoxidase activity were noted. As well as, reduction in MCP-1 and collagen type-1 levels along with Hedgehog signaling down-regulating expression in such cells, Patched-1, Smoothened, and GLi-1. Conclusion: The potent bioactive therapeutic Icariin combined with MSCs induces a significantly greater improvement, compared to each therapy alone.

Hedgehog Signaling and Truncated GLI1 in Cancer

The hedgehog (HH) signaling pathway regulates normal cell growth and differentiation. As a consequence of improper control, aberrant HH signaling results in tumorigenesis and supports aggressive phenotypes of human cancers, such as neoplastic transformation, tumor progression, metastasis, and drug resistance. Canonical activation of HH signaling occurs through binding of HH ligands to the transmembrane receptor Patched 1 (PTCH1), which derepresses the transmembrane G protein-coupled receptor Smoothened (SMO). Consequently, the glioma-associated oncogene homolog 1 (GLI1) zinc-finger transcription factors, the terminal effectors of the HH pathway, are released from suppressor of fused (SUFU)-mediated cytoplasmic sequestration, permitting nuclear translocation and activation of target genes. Aberrant activation of this pathway has been implicated in several cancer types, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, glioblastoma, and cancers of lung, colon, stomach, pancreas, ovarian, and breast. Therefore, several components of the HH pathway are under investigation for targeted cancer therapy, particularly GLI1 and SMO. GLI1 transcripts are reported to undergo alternative splicing to produce truncated variants: loss-of-function GLI1ΔN and gain-of-function truncated GLI1 (tGLI1). This review covers the biochemical steps necessary for propagation of the HH activating signal and the involvement of aberrant HH signaling in human cancers, with a highlight on the tumor-specific gain-of-function tGLI1 isoform.