Porcupine (PORCN) is a membrane-bound protein of the endoplasmic reticulum, which modifies Wnt proteins by adding palmitoleic acid. This modification is essential for Wnt ligand secretion. Patients with mutated PORCN display various skeletal abnormalities likely stemming from disrupted Wnt signaling pathways during the chondrocyte differentiation. To uncover the mechanism of PORCN action during chondrogenesis, we used 2 different PORCN inhibitors, C59 and LGK974, in several model systems, including micromasses, 3D cell cultures, long bone tissue cultures, and zebrafish animal model. PORCN inhibitors enhanced cartilaginous extracellular matrix (ECM) production and accelerated chondrocyte differentiation, which resulted in the earlier induction of cellular hypertrophy as well as cartilaginous mass expansion in micromass cultures and cartilaginous organoids. In addition, both PORCN inhibitors expanded the hypertrophic zone and reduced the proliferative zone in the growth plate. This led to a significant increase in cartilaginous tissue and ultimately resulted in the elongation of tibias in the mouse organ cultures. Also, LGK974 treatment of Danio rerio embryos induced expansion of craniofacial cartilage width together with the shortening of the body axis, which was consistent with a phenomenon occurring upon inhibition of non-canonical Wnt signaling. By combining PORCN inhibition with exogenous Wnt proteins activating either canonical/β-catenin (WNT3a) or non-canonical (WNT5a) signaling, we propose that the key mechanism mediating pro-chondrogenic effects of PORCN inhibition is the removal of canonical ligands that prevent chondrocyte differentiation. In summary, our results provide evidence of the distinct role of PORCN in both the early and late stages of cartilage development. Further, our data demonstrate that PORCN inhibitors can be used in the experimental and clinical strategies that need to trigger chondrocyte differentiation and/or cartilage outgrowth.

• Klíčová slova
• Wnt, cartilage, chondrogenesis, hypertrophy, jaw hypoplasia, micromass cultures, orofacial anomalies, porcupine, tibia,
• Publikační typ
• časopisecké články MeSH

Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/β-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of β-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/β-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate β-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/β KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-β-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/β-catenin pathway at the level of β-catenin and Axin.

• Klíčová slova
• Axin, NanoBRET, Wnt pathway, alternative splicing, casein kinase 1 alpha (CK1α), casein kinase 1 alpha-like (CK1α-like), gene knockout, inhibitor, phosphorylation, β-catenin,
• MeSH
• alternativní sestřih MeSH
• beta-katenin * metabolismus   genetika   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• kaseinkinasa Ialfa * metabolismus   genetika   MeSH
• kinasa 3 glykogensynthasy metabolismus   genetika   MeSH
• kinasa glykogensynthasy 3beta metabolismus   genetika   MeSH
• lidé MeSH
• protein Wnt3A metabolismus   genetika   MeSH
• signální dráha Wnt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• beta-katenin * MeSH
• kaseinkinasa Ialfa * MeSH
• kinasa 3 glykogensynthasy MeSH
• kinasa glykogensynthasy 3beta MeSH
• protein Wnt3A MeSH
• WNT3A protein, human MeSH Prohlížeč

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by stem cells residing at the crypt bottom. Paneth cells form a major element of the niche microenvironment providing various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. Different Wnt ligands can selectively activate β-catenin-dependent (canonical) or -independent (noncanonical) signaling. Here, we report that the Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and noncanonical Wnt (Wnt/PCP) signaling. Daam1/2 interacts with the Wnt inhibitor RNF43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing RNF43-dependent degradation of the Wnt receptor, Frizzled (Fzd). Single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion because of defective Wnt/PCP signaling. Together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and noncanonical Wnt, which is fundamental for specifying an adequate number of Paneth cells.

• MeSH
• buněčná diferenciace MeSH
• kmenové buňky metabolismus   MeSH
• Panethovy buňky * MeSH
• savci MeSH
• signální dráha Wnt * MeSH
• střeva MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The migratory properties of leukemic cells are commonly associated with their pathological potential and can significantly affect the disease progression. While the research in immunopathology mostly employed powerful indirect methods such as flow cytometry, these cells were rarely observed directly using live imaging microscopy. This is especially true for the malignant cells of the B-cell lineage, such as those originating from chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In this study, we employed open-source image analysis tools to automatically and quantitatively describe the amoeboid migration of four B-cell leukemic and lymphoma cell lines and primary CLL cells. To avoid the effect of the shear stress of the medium on these usually non-adherent cells, we have confined the cells using a modified under-agarose assay. Surprisingly, the behavior of tested cell lines differed substantially in terms of basal motility or response to chemokines and VCAM1 stimulation. Since casein kinase 1 (CK1) was reported as a regulator of B-cell migration and a promoter of CLL, we looked at the effects of CK1 inhibition in more detail. Migration analysis revealed that CK1 inhibition induced rapid negative effects on the migratory polarity of these cells, which was quantitatively and morphologically distinct from the effect of ROCK inhibition. We have set up an assay that visualizes endocytic vesicles in the uropod and facilitates morphological analysis. This assay hints that the effect of CK1 inhibition might be connected to defects in polarized intracellular transport. In summary, 1) we introduce and validate a pipeline for the imaging and quantitative assessment of the amoeboid migration of CLL/MCL cells, 2) we provide evidence that the assay is sensitive enough to mechanistically study migration defects identified by the transwell assay, and 3) we describe the polarity defects induced by inhibition or deletion of CK1ε.

• Klíčová slova
• B cells, amoeboid cell migration, casein kinase 1, chronic lymphocytic leukemia, live imaging, mantle cell lymphoma, uropod,
• Publikační typ
• časopisecké články MeSH