OBJECTIVE: Despite increasing knowledge regarding the cellular and molecular mechanisms of liver fibrogenesis, there is currently no approved drug for the treatment of liver fibrosis. Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing an attractive therapeutic tool for tissue damage and inflammation. This study was designed to determine the protective effect and underlying mechanism of human umbilical cord-derived MSCs (UC-MSCs) on thioacetamide-induced liver fibrosis.
METHODS: Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide (TAA). Some mice were then given injection of UC-MSCs or UC-MSCs-derived exosomes (UC-MSCs-Exo) via the tail vein. Liver tissues were collected for histologic analysis.
RESULTS: We found that administration of UC-MSCs significantly reduced serum alanine aminotransferase and aspartate aminotransferase levels, and attenuated hepatic inflammation and fibrosis. Moreover, the therapeutic effect of UC-MSCs-derived exosomes was similar to that of UC-MSCs. Intriguingly, UC-MSCs-Exo treatment downregulated the expression of smoothened (SMO), a fundamental component of Hedgehog signaling which plays a critical role in fibrogenesis, and subsequently inhibited the activation of hepatic stellate cells, a central driver of fibrosis in experimental and human liver injury. Furthermore, the anti-inflammatory and anti-fibrotic effects of UCMSCs- Exo was reversed by the SMO agonist SAG treatment in mice.
CONCLUSIONS: Our findings suggest that UC-MSCs-Exo exert therapeutic effects on liver fibrosis, at least in part, through inhibiting the Hedgehog/SMO signaling pathway.

Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEFs) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia, and upregulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild-type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. The CILK1 A615T mutant protein was not increased to the same level as the wild-type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single-residue variant of CILK1 is compromised, and this impairs the maintenance of primary cilia and Hedgehog signaling.

Identification of potential bacterial players in colorectal tumorigenesis has been a focus of intense research. Herein, we find that Clostridium symbiosum (C. symbiosum) is selectively enriched in tumor tissues of patients with colorectal cancer (CRC) and associated with higher colorectal adenoma recurrence after endoscopic polypectomy. The tumorigenic effect of C. symbiosum is observed in multiple murine models. Single-cell transcriptome profiling along with functional assays demonstrates that C. symbiosum promotes the proliferation of colonic stem cells and enhances cancer stemness. Mechanistically, C. symbiosum intensifies cellular cholesterol synthesis by producing branched-chain amino acids (BCAAs), which sequentially activates Sonic hedgehog signaling. Low dietary BCAA intake or blockade of cholesterol synthesis by statins could partially abrogate the C. symbiosum-induced cell proliferation in vivo and in vitro. Collectively, we reveal C. symbiosum as a bacterial driver of colorectal tumorigenesis, thus identifying a potential target in CRC prediction, prevention, and treatment.

Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson\'s and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson\'s. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson\'s.

The functional maturation of the pituitary gland requires adequate cell differentiation and vascular network formation. Although spatiotemporal signaling and transcription factors are known to govern pituitary development, the involvement of primary cilia, nonmoving hair-like organelles, remains unclear. In this study, we uncovered the contribution of primary cilia to cell-type determination and vascular network formation during pituitary development. Homozygous knockout mice lacking a ciliary kinase, Dyrk2-/-, exhibit abnormalities in ciliary structure and pituitary hypoplasia, accompanied by varying degrees of failure in differentiation among all types of hormone-producing cells in the anterior lobe. Aberrations in cell differentiation in Dyrk2-/- mice arise from a decrease in the expression of crucial transcription factors, Lhx4, Lhx3, and Prop1, resulting from the inactivity of Hedgehog (Hh) signaling during the early stages of development. Furthermore, the loss of Dyrk2 results in vascular system abnormalities during the middle to late stages of development. Mechanistically, transcriptome analyses revealed the downregulation of vitronectin-integrin αvβ3-VEGFR2 signaling, essential for orchestrating vascular development. Collectively, our findings demonstrate that primary cilia play a pivotal role as critical regulators of cell survival, cell determination, and angiogenesis during pituitary gland development through the activation of Hh signaling. These findings expand our understanding of the potential link between pituitary dysfunction in human disorders and ciliopathies.

The gene encoding the NUAK family kinase 1 (NUAK1) is frequently amplified and its expression is upregulated, activating oncogenic signaling in various cancers. However, little is known about its role in gastric cancer (GC). We investigate the mechanistic links among NUAK1, Hedgehog signaling, and tumorigenesis in GC. NUAK1 overexpression is validated in local and public GC cohorts. Patient-derived xenograft and transgenic mouse models demonstrate that NUAK1 depletion or inhibition dramatically ameliorates gastric tumorigenesis. NUAK1 upregulates GLI1 expression by activating STAT5-mediated transcription and stabilizing GLI1 protein. NUAK1 depletion or inhibition impairs cancer cell expansion, tumor formation, and chemotherapy resistance in in vitro and in vivo models. Clinicopathological analysis confirms that upregulated NUAK1 expression correlates with poor prognosis and chemotherapy resistance in human GC. Our findings demonstrate that the signaling axis NUAK1/STAT5/GLI1 promotes cancer cell expansion and tumorigenesis and indicate that NUAK1 is an attractive therapeutic target and prognostic factor in GC.

The development of amoebic liver abscess (ALA) leads to liver necrosis, accompanied by an exacerbated inflammatory response and the formation of multiple granulomas. Adequate management of the infection through the administration of treatment and the timely response of the organ to the damage allows the injury to heal with optimal regeneration without leaving scar tissue, which does not occur in other types of damage such as viral hepatitis that may conducts to fibrosis or cirrhosis. The Hedgehog signaling pathway (Hh) is crucial in the embryonic stage, while in adults it is usually reactivated in response to acute or chronic injuries, regeneration, and wound healing. In this work, we characterized Hh in experimental hepatic amoebiasis model, with the administration of treatment with metronidazole, as well as a pathway inhibitor (cyclopamine), through histological and immunohistochemical analyses including an ultrastructure analysis through transmission electron microscopy. The results showed an increase in the percentage of lesions obtained, a decrease in the presence of newly formed hepatocytes, a generalized inflammatory response, irregular distribution of type I collagen accompanied by the presence of fibroblast-type cells and a decrease in effector cells of this pathway. These results constitute the first evidence of the association of the activation of Hh with the liver regeneration process in experimental amebiasis.

BACKGROUND: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown.
METHODS: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling.
RESULTS: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force.
CONCLUSIONS: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

Exposure to inorganic arsenic (As) is recognized as a risk factor for non-melanoma skin cancer (NMSC). We followed up with 7000 adults for 6 years who were exposed to As. During follow-up, 2.2% of the males and 1.3% of the females developed basal cell carcinoma (BCC), while 0.4% of the male and 0.2% of the female participants developed squamous cell carcinoma (SCC). Using a panel of more than 400 cancer-related genes, we detected somatic mutations (SMs) in the first 32 NMSC samples (BCC = 26 and SCC = 6) by comparing paired (tissue-blood) samples from the same individual and then comparing them to the SM in healthy skin tissue from 16 participants. We identified (a) a list of NMSC-associated SMs, (b) SMs present in both NMSC and healthy skin, and (c) SMs found only in healthy skin. We also demonstrate that the presence of non-synonymous SMs in the top mutated genes (like PTCH1, NOTCH1, SYNE1, PKHD1 in BCC and TP53 in SCC) significantly affects the magnitude of differential expressions of major genes and gene pathways (basal cell carcinoma pathways, NOTCH signaling, IL-17 signaling, p53 signaling, Wnt signaling pathway). These findings may help select groups of patients for targeted therapy, like hedgehog signaling inhibitors, IL17 inhibitors, etc., in the future.