Mebendazole preferentially inhibits cilia formation and exerts anticancer activity by synergistically augmenting DNA damage.

The cilium is a microtubule-based organelle that plays a pivotal role in embryonic development and maintenance of physiological functions in the human body. In addition to their function as sensors that transduce diverse extracellular signals, including growth factors, fluid flow, and physical forces, cilia are intricately involved in cell cycle regulation and preservation of DNA integrity, as their formation and resorption dynamics are tightly linked to cell cycle progression. Recently, several studies have linked defects in specific ciliary proteins to the DNA damage response. However, it remains unclear whether and how primary cilia contribute to cancer development. Mebendazole (MBZ) is an anthelmintic drug with anticancer properties in some cancer cells. MBZ is continuously being tested for clinical studies, but the precise mechanism of its anticancer activities remains unknown. Here, using Xenopus laevis embryos as a model system, we discovered that MBZ significantly hinders cilia formation and induces DNA damage. Remarkably, primary cilium-bearing cancer cells exhibited heightened vulnerability to combined treatment with MBZ and conventional anticancer drugs. Our findings shed light on the specific influence of MBZ on cilia, rather than cytosolic microtubules, in triggering DNA damage, elucidating a previously unidentified mechanism underlying potential MBZ-mediated cancer therapy.

Ckb and Ybx2 interact with Ribc2 and are necessary for the ciliary beating of multi-cilia.

BACKGROUND: Motile cilia in a vertebrate are important to sustaining activities of life. Fluid flow on the apical surface of several tissues, including bronchial epithelium, ependymal epithelium, and fallopian tubules is generated by the ciliary beating of motile cilia. Multi-ciliated cells in ependymal tissue are responsible for the circulation of cerebrospinal fluid (CSF), which is essential for the development and homeostasis of the central nervous system, and airway tissues are protected from external contaminants by cilia-driven mucosal flow over the top of the airway epithelium. OBJECTIVE: A previous study reported that reduction of Ribc2 protein leads to disruption of ciliary beating in multi-ciliated cells. However, knowledge regarding the molecular function of Ribc2 is limited, thus currently available information is also limited. Therefore, we evaluated the importance of proteins involved in the interaction with Ribc2 in the process of ciliary beating. METHODS: Immunoprecipitation and mass spectrometry analysis was performed for the discovery of proteins involved in the interaction with Ribc2. Expression of the target gene was inhibited by injection of antisense morpholinos and measurement of the fluid flow on the embryonic epidermis of Xenopus was performed using fluorescent beads for examination of the ciliary beating of multi cilia. In addition, the flag-tagged protein was expressed by injection of mRNA and the changes in protein localization in the cilia were measured by immunostaining and western blot analysis for analysis of the molecular interaction between Ribc2 and Ribc2 binding proteins in multi-cilia. RESULTS: The IP/MS analysis identified Ckb and Ybx2 as Ribc2 binding proteins and our results showed that localization of both Ckb and Ybx2 occurs at the axoneme of multi-cilia on the embryonic epithelium of Xenopus laevis. In addition, our findings confirmed that knock-down of Ckb or Ybx2 resulted in abnormal ciliary beating and reduction of cilia-driven fluid flow on multi-cilia of Xenopus laevis. In addition, significantly decreased localization of Ckb or Ybx2 in the ciliary axoneme was observed in Ribc2-depleted multi-cilia. CONCLUSION: Ckb and Ybx2 are involved in the interaction with Ribc2 and are necessary for the ciliary beating of multi-cilia.

GJA1 depletion causes ciliary defects by affecting Rab11 trafficking to the ciliary base.

The gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein α1 (GJA1) is the most commonly expressed subunit. A recent study showed that GJA1 is necessary for the maintenance of motile cilia; however, the molecular mechanism and function of GJA1 in ciliogenesis remain unknown. Here, we examined the functions of GJA1 during ciliogenesis in human retinal pigment epithelium-1 and Xenopus laevis embryonic multiciliated-cells. GJA1 localizes to the motile ciliary axonemes or pericentriolar regions beneath the primary cilium. GJA1 depletion caused malformation of both the primary cilium and motile cilia. Further study revealed that GJA1 depletion affected several ciliary proteins such as BBS4, CP110, and Rab11 in the pericentriolar region and basal body. Interestingly, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, a key regulator during ciliogenesis, was immunoprecipitated with GJA1 and GJA1 knockdown caused the mislocalization of Rab11. These findings suggest that GJA1 regulates ciliogenesis by interacting with the Rab11-Rab8 ciliary trafficking pathway.

Augmented ERAD (ER-associated degradation) activity in chondrocytes is necessary for cartilage development and maintenance.

Chondrocytes secrete massive extracellular matrix (ECM) molecules that are produced, folded, and modified in the endoplasmic reticulum (ER). Thus, the ER-associated degradation (ERAD) complex-which removes misfolded and unfolded proteins to maintain proteostasis in the ER- plays an indispensable role in building and maintaining cartilage. Here, we examined the necessity of the ERAD complex in chondrocytes for cartilage formation and maintenance. We show that ERAD gene expression is exponentially increased during chondrogenesis, and disruption of ERAD function causes severe chondrodysplasia in developing embryos and loss of adult articular cartilage. ERAD complex malfunction also causes abnormal accumulation of cartilage ECM molecules and subsequent chondrodysplasia. ERAD gene expression is decreased in damaged cartilage from patients with osteoarthritis (OA), and disruption of ERAD function in articular cartilage leads to cartilage destruction in a mouse OA model.

Integrin ß-like 1 protein (ITGBL1) promotes cell migration by preferentially inhibiting integrin-ECM binding at the trailing edge.

BACKGROUND: Cell migration is a basic cellular behavior involved in multiple phenomena in the human body such as embryonic development, wound healing, immune reactions, and cancer metastasis. For proper cell migration, integrin and the ECM binding complex must be disassembled for the retraction of trailing edges. OBJECTIVE: Integrin must be differentially regulated at leading edges or trailing edges during cell migration. Previously, we showed that ITGBL1 was a secreted protein and inhibits integrin activity. Therefore, we examined the function of ITGBL1 on the retraction of trailing edges during cell migration. METHODS: To examined the function of ITGBL1 on cell migration, we knocked-down or overexpressed ITGBL1 by using ITGBL1 siRNA or ITGBL1 plasmid DNA in human chondrocytes or ATDC5 cells. We then characterized cellular migration and directionality by performing wound healing assays. Also, to analyze leading-edge formation and trailing-edge retraction, we labeled cell membranes with membrane-GFP and performed live imaging of migrating cells and. Finally, we specifically detected active forms of integrin, FAK and Vinculin using specific antibodies upon ITGBL1 depletion or overexpression. RESULT: In this study, ITGBL1 preferentially inhibited integrin activity at the trailing edges to promote cell migration. ITGBL1-depleted cells showed increased focal adhesions at the membranous traces of trailing edges to prevent the retraction of trailing edges. In contrast, overexpression of ITGBL1 upregulated directional cell migration by promoting focal adhesion disassembly at the trailing edges. CONCLUSION: ITGBL1 facilitates directional cell migration by promoting disassembly of the trailing edge focal adhesion complex.

Extracellular matrixes and neuroinflammation.

The extracellular matrix is a critical component of every human tissue. ECM not only functions as a structural component but also regulates a variety of cellular processes such as cell migration, differentiation, proliferation, and cell death. In addition, current studies suggest that ECM is critical for the pathophysiology of various human diseases. ECM is composed of diverse components including several proteins and polysaccharide chains such as chondroitin sulfate, heparan sulfate, and hyaluronic acid. Each component of ECM exerts its own functions in cellular and pathophysiological processes. One of the interesting recent findings is that ECM is involved in inflammatory responses in various human tissues. In this review, we summarized the known functions of ECM in neuroinflammation after acute injury and chronic inflammatory diseases of the central nerve systems. [BMB Reports 2020; 53(10): 491-499].

Peroxiredoxin5 Controls Vertebrate Ciliogenesis by Modulating Mitochondrial Reactive Oxygen Species.

AIMS: Peroxiredoxin5 (Prdx5), a thioredoxin peroxidase, is an antioxidant enzyme that is widely studied for its antioxidant properties and protective roles in neurological and cardiovascular disorders. This study is aimed at investigating the functional significance of Prdx5 in mitochondria and at analyzing its roles in ciliogenesis during the process of vertebrate development. RESULTS: We found that several Prdx genes were strongly expressed in multiciliated cells in developing Xenopus embryos, and their peroxidatic functions were crucial for normal cilia development. Depletion of Prdx5 increased levels of cellular reactive oxygen species (ROS), consequently leading to mitochondrial dysfunction and abnormal cilia formation. Proteomic and transcriptomic approaches revealed that excessive ROS accumulation on Prdx5 depletion subsequently reduced the expression level of pyruvate kinase (PK), a key metabolic enzyme in energy production. We further confirmed that the promotor activity of PK was significantly reduced on Prdx5 depletion and that the reduction in PK expression and its promoter activity led to ciliary defects observed in Prdx5-depleted cells. INNOVATION: Our data revealed the novel relationship between ROS and Prdx5 and the consequent effects of this interaction on vertebrate ciliogenesis. The normal process of ciliogenesis is interrupted by the Prdx5 depletion, resulting in excessive ROS levels and suggesting cilia as vulnerable targets of ROS. CONCLUSION: Prdx5 plays protective roles in mitochondria and is critical for normal cilia development by regulating the levels of ROS. The loss of Prdx5 is associated with excessive production of ROS, resulting in mitochondrial dysfunction and aberrant ciliogenesis.

ITGBL1 modulates integrin activity to promote cartilage formation and protect against arthritis.

Developing and mature chondrocytes constantly interact with and remodel the surrounding extracellular matrix (ECM). Recent research indicates that integrin-ECM interaction is differentially regulated during cartilage formation (chondrogenesis). Integrin signaling is also a key source of the catabolic reactions responsible for joint destruction in both rheumatoid arthritis and osteoarthritis. However, we do not understand how chondrocytes dynamically regulate integrin signaling in such an ECM-rich environment. Here, we found that developing chondrocytes express integrin-ß-like 1 (Itgbl1) at specific stages, inhibiting integrin signaling and promoting chondrogenesis. Unlike cytosolic integrin inhibitors, ITGBL1 is secreted and physically interacts with integrins to down-regulate activity. We observed that Itgbl1 expression was strongly reduced in the damaged articular cartilage of patients with osteoarthritis (OA). Ectopic expression of Itgbl1 protected joint cartilage against OA development in the destabilization of the medial meniscus-induced OA mouse model. Our results reveal ITGBL1 signaling as an underlying mechanism of protection against destructive cartilage disorders and suggest the potential therapeutic utility of targeting ITGBL1 to modulate integrin signaling in human disease.

A thioredoxin fold protein Sh3bgr regulates Enah and is necessary for proper sarcomere formation.

The sh3bgr (SH3 domain binding glutamate-rich) gene encodes a small protein containing a thioredoxin-like fold, SH3 binding domain, and glutamate-rich domain. Originally, it was suggested that increased expression of Sh3bgr may cause the cardiac phenotypes in Down's syndrome. However, it was recently reported that the overexpression of Sh3bgr did not cause any disease phenotypes in mice. In this study, we have discovered that Sh3bgr is critical for sarcomere formation in striated muscle tissues and also for heart development. Sh3bgr is strongly expressed in the developing somites and heart in Xenopus. Morpholino mediated-knockdown of sh3bgr caused severe malformation of heart tissue and disrupted segmentation of the somites. Further analysis revealed that Sh3bgr specifically localized to the Z-line in mature sarcomeres and that knockdown of Sh3bgr completely disrupted sarcomere formation in the somites. Moreover, overexpression of Sh3bgr resulted in abnormally discontinues thick firmaments in the somitic sarcomeres. We suggest that Sh3bgr does its function at least partly by regulating localization of Enah for the sarcomere formation. In addition, we provide the data supporting Sh3bgr is also necessary for proper heart development in part by affecting the Enah protein level.