KIF11 UFMylation Maintains Photoreceptor Cilium Integrity and Retinal Homeostasis.

The photoreceptor cilium is vital for maintaining the structure and function of the retina. However, the molecular mechanisms underlying the photoreceptor cilium integrity and retinal homeostasis are largely unknown. Herein, it is shown that kinesin family member 11 (KIF11) localizes at the transition zone (connecting cilium) of the photoreceptor and plays a crucial role in orchestrating the cilium integrity. KIF11 depletion causes malformations of both the photoreceptor ciliary axoneme and membranous discs, resulting in photoreceptor degeneration and the accumulation of drusen-like deposits throughout the retina. Mechanistic studies show that the stability of KIF11 is regulated by an interplay between its UFMylation and ubiquitination; UFMylation of KIF11 at lysine 953 inhibits its ubiquitination by synoviolin 1 and thereby prevents its proteasomal degradation. The lysine 953-to-arginine mutant of KIF11 is more stable than wild-type KIF11 and also more effective in reversing the ciliary and retinal defects induced by KIF11 depletion. These findings identify a critical role for KIF11 UFMylation in the maintenance of photoreceptor cilium integrity and retinal homeostasis.

High LGALS3 expression induced by HCP5/hsa-miR-27b-3p correlates with poor prognosis and tumor immune infiltration in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is widely recognized for its unfavorable prognosis. Increasing evidence has revealed that LGALS3 has an essential function in initiating and developing several malignancies in humans. Nevertheless, thorough analysis of the expression profile, clinical prognosis, pathway prediction, and immune infiltration of LGALS3 has not been fully explored in HCC. METHODS: In this study, an initial pan-cancer analysis was conducted to investigate the expression and prognosis of LGALS3. Following a comprehensive analysis, which included expression analysis and correlation analysis, noncoding RNAs that contribute to the overexpression of LGALS3 were subsequently identified. This identification was further validated using HCC clinical tissue samples. TIMER2 and GEPIA2 were employed to examine the correlation between LGALS3 and HCP5 with immunological checkpoints, cell chemotaxis, and immune infiltration in HCC. The R program was applied to analyze the expression distribution of immune score in in HCC patients with high and low LGALS3 expression. The expression profiles of immune checkpoints were also analyzed. Use R to perform GSVA analysis in order to explore potential signaling pathways. RESULTS: First, we conducted pan-cancer analysis for LGALS3 expression level through an in-depth analysis of public databases and found that HCC has a high LGALS3 gene and protein expression level, which were then verified in clinical HCC specimens. Meanwhile, high LGALS3 gene expression is related to malignant progression and poor prognosis of HCC. Univariate and multivariate analyses confirmed that LGALS3 could serve as an independent prognostic marker for HCC. Next, by combining comprehensive analysis and validation on HCC clinical tissue samples, we hypothesize that the HCP5/hsa-miR-27b-3p axis could serve as the most promising LGALS3 regulation mechanism in HCC. KEGG and GO analyses highlighted that the LGALS3-related genes were involved in tumor immunity. Furthermore, we detected a significant positive association between LGALS3 and HCP5 with immunological checkpoints, cell chemotaxis, and immune infiltration. In addition, high LGALS3 expression groups had significantly higher immune cell scores and immune checkpoint expression levels. Finally, GSVA analysis was performed to predict potential signaling pathways linked to LGALS3 and HCP5 in immune evasion and metabolic reprogramming of HCC. CONCLUSIONS: Our findings indicated that the upregulation of LGALS3 via the HCP5/hsa-miR-27b-3p axis is associated with unfavorable prognosis and increased tumor immune infiltration in HCC.

Dynamic changes of endothelial and stromal cilia are required for the maintenance of corneal homeostasis.

Primary cilia are distributed extensively within the corneal epithelium and endothelium. However, the presence of cilia in the corneal stroma and the dynamic changes and roles of endothelial and stromal cilia in corneal homeostasis remain largely unknown. Here, we present compelling evidence for the presence of primary cilia in the corneal stroma, both in vivo and in vitro. We also demonstrate dynamic changes of both endothelial and stromal cilia during corneal development. In addition, our data show that cryoinjury triggers dramatic cilium formation in the corneal endothelium and stroma. Furthermore, depletion of cilia in mutant mice lacking intraflagellar transport protein 88 compromises the corneal endothelial capacity to establish the effective tissue barrier, leading to an upregulation of α-smooth muscle actin within the corneal stroma in response to cryoinjury. These observations underscore the essential involvement of corneal endothelial and stromal cilia in maintaining corneal homeostasis and provide an innovative strategy for the treatment of corneal injuries and diseases.

XIAP-mediated degradation of IFT88 disrupts HSC cilia to stimulate HSC activation and liver fibrosis.

Activation of hepatic stellate cells (HSCs) plays a critical role in liver fibrosis. However, the molecular basis for HSC activation remains poorly understood. Herein, we demonstrate that primary cilia are present on quiescent HSCs but exhibit a significant loss upon HSC activation which correlates with decreased levels of the ciliary protein intraflagellar transport 88 (IFT88). Ift88-knockout mice are more susceptible to chronic carbon tetrachloride-induced liver fibrosis. Mechanistic studies show that the X-linked inhibitor of apoptosis (XIAP) functions as an E3 ubiquitin ligase for IFT88. Transforming growth factor-ß (TGF-ß), a profibrotic factor, enhances XIAP-mediated ubiquitination of IFT88, promoting its proteasomal degradation. Blocking XIAP-mediated IFT88 degradation ablates TGF-ß-induced HSC activation and liver fibrosis. These findings reveal a previously unrecognized role for ciliary homeostasis in regulating HSC activation and identify the XIAP-IFT88 axis as a potential therapeutic target for liver fibrosis.

Pathologically relevant aldoses and environmental aldehydes cause cilium disassembly via formyl group-mediated mechanisms.

Carbohydrate metabolism disorders (CMDs), such as diabetes, galactosemia, and mannosidosis, cause ciliopathy-like multiorgan defects. However, the mechanistic link of cilia to CMD complications is still poorly understood. Herein, we describe a significant cilium disassembly upon treatment of cells with pathologically relevant aldoses rather than the corresponding sugar alcohols. Moreover, environmental aldehydes are able to trigger cilium disassembly by the steric hindrance effect of their formyl groups. Mechanistic studies reveal that aldehydes stimulate extracellular calcium influx across the plasma membrane, which subsequently activates the calmodulin-Aurora A-histone deacetylase 6 pathway to deacetylate axonemal microtubules and triggers cilium disassembly. In vivo experiments further show that Hdac6 knockout mice are resistant to aldehyde-induced disassembly of tracheal cilia and sperm flagella. These findings reveal a previously unrecognized role for formyl group-mediated cilium disassembly in the complications of CMDs.

O-GlcNAcylation Regulates Centrosome Behavior and Cell Polarity to Reduce Pulmonary Fibrosis and Maintain the Epithelial Phenotype.

O-GlcNAcylation functions as a cellular nutrient and stress sensor and participates in almost all cellular processes. However, it remains unclear whether O-GlcNAcylation plays a role in the establishment and maintenance of cell polarity, because mice lacking O-GlcNAc transferase (OGT) are embryonically lethal. Here, a mild Ogt knockout mouse model is constructed and the important role of O-GlcNAcylation in establishing and maintaining cell polarity is demonstrated. Ogt knockout leads to severe pulmonary fibrosis and dramatically promotes epithelial-to-mesenchymal transition. Mechanistic studies reveal that OGT interacts with pericentriolar material 1 (PCM1) and centrosomal protein 131 (CEP131), components of centriolar satellites required for anchoring microtubules to the centrosome. These data further show that O-GlcNAcylation of PCM1 and CEP131 promotes their centrosomal localization through phase separation. Decrease in O-GlcNAcylation prevents PCM1 and CEP131 from localizing to the centrosome, instead dispersing these proteins throughout the cell and impairing the microtubule-centrosome interaction to disrupt centrosome positioning and cell polarity. These findings identify a previously unrecognized role for protein O-GlcNAcylation in establishing and maintaining cell polarity with important implications for the pathogenesis of pulmonary fibrosis.

Primary cilium-mediated signaling cascade suppresses age-related biliary fibrosis.

The primary cilium is increasingly recognized as a crucial player in the physiology of biliary epithelial cells (BECs). However, the precise role of primary cilia in the development of age-related biliary fibrosis remains unclear. Herein, using cilium-deficient mice, we demonstrate that disruption of ciliary homeostasis in BECs in aged mice leads to significant bile duct proliferation, augmented biliary fibrosis, and heightened indicators of liver injury. Our RNA-sequencing data revealed a dysregulation in genes associated with various biological processes such as bile secretion, fatty acid metabolism, and inflammation. Loss of primary cilia also significantly enhanced signaling pathways driving the development of biliary fibrosis. Our findings collectively suggest that loss of primary cilia in the BECs of aged mice initiates a cascade of signaling events that contribute to biliary fibrosis, highlighting the primary cilium as a potential therapeutic target in the treatment of fibrosing cholangiopathies.

HDAC6 deacetylates IDH1 to promote the homeostasis of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are cells mainly present in the bone marrow and capable of forming mature blood cells. However, the epigenetic mechanisms governing the homeostasis of HSPCs remain elusive. Here, we demonstrate an important role for histone deacetylase 6 (HDAC6) in regulating this process. Our data show that the percentage of HSPCs in Hdac6 knockout mice is lower than in wild-type mice due to decreased HSPC proliferation. HDAC6 interacts with isocitrate dehydrogenase 1 (IDH1) and deacetylates IDH1 at lysine 233. The deacetylation of IDH1 inhibits its catalytic activity and thereby decreases the 5-hydroxymethylcytosine level of ten-eleven translocation 2 (TET2) target genes, changing gene expression patterns to promote the proliferation of HSPCs. These findings uncover a role for HDAC6 and IDH1 in regulating the homeostasis of HSPCs and may have implications for the treatment of hematological diseases.

Src inhibition induces mitotic arrest associated with chromosomal passenger complex.

The non-receptor tyrosine kinase Src plays a key role in cell division, migration, adhesion, and survival. Src is overactivated in several cancers, where it transmits signals that promote cell survival, mitosis, and other important cancer hallmarks. Src is therefore a promising target in cancer therapy, but the underlying mechanisms are still uncertain. Here we show that Src is highly conserved across different species. Src expression increases during mitosis and is localized to the chromosomal passenger complex. Knockdown or inhibition of Src induces multipolar spindle formation, resulting in abnormal expression of the Aurora B and INCENP components of the chromosomal passenger complex. Molecular mechanism studies have found that Src interacts with and phosphorylates INCENP. This then leads to incorrect chromosome arrangement and segregation, resulting in cell division failure. Herein, Src and chromosomal passenger complex co-localize and Src inhibition impedes mitotic progression by inducing multipolar spindle formation. These findings provide novel insights into the molecular basis for using Src inhibitors to treat cancer.

CYLD inhibits osteoclastogenesis to ameliorate alveolar bone loss in mice with periodontitis.

Periodontitis is a chronic immune inflammatory disease that can lead to the destruction and loss of the tooth-supporting apparatus. During this process, the balance between bone absorption mediated by osteoclasts and bone formation mediated by osteoblasts is damaged. Consistent with previous studies, we observed that depletion of cylindromatosis (CYLD) resulted in an osteoporotic bone phenotype. However, the effect of CYLD deficiency on periodontitis is undetermined. Here, we investigated whether CYLD affects periodontal tissue homeostasis in experimental periodontitis in Cyld knockout (KO) mice, and we explored the underlying mechanisms. Interestingly, we discovered significant alveolar bone density loss and severely reduced alveolar bone height in Cyld KO mice with experimentally induced periodontitis. We observed increased osteoclast number and activity in both the femurs and alveolar bones, accompanied by the downregulation of osteogenesis genes and upregulation of osteoclastogenesis genes of alveolar bones in ligatured Cyld KO mice. Taken together, our findings demonstrate that the deletion of CYLD in mice plays a vital role in the pathogenesis of periodontal bone loss and suggest that CYLD might exert an ameliorative effect on periodontal inflammatory responses.

Regulation of epidermal stratification and development by basal keratinocytes.

The epidermis is a stratified squamous epithelium distributed in the outermost layer of the skin and is intimately involved in the formation of a physical barrier to pathogens. Basal keratinocytes possess the properties of stem cells and play an essential role in epidermal development and skin damage recovery. Therefore, understanding the molecular mechanism of how basal keratinocytes participate in epidermal development and stratification is vital for preventing and treating skin lesions. During epidermal morphogenesis, the symmetric division of basal keratinocytes contributes to the extension of skin tissues, while their asymmetric division and migration facilitate epidermal stratification. In this review, we summarize the process of epidermal stratification and illustrate the molecular mechanisms underlying epidermal morphogenesis. Furthermore, we discuss the coordination of multiple signaling pathways and transcription factors in epidermal stratification, together with the roles of cell polarity and cell dynamics during the process.

CYLD deubiquitinates plakoglobin to promote Cx43 membrane targeting and gap junction assembly in the heart.

During heart maturation, gap junctions assemble into hemichannels and polarize to the intercalated disc at cell borders to mediate electrical impulse conduction. However, the molecular mechanism underpinning cardiac gap junction assembly remains elusive. Herein, we demonstrate an important role for the deubiquitinating enzyme cylindromatosis (CYLD) in this process. Depletion of CYLD in mice impairs the formation of cardiac gap junctions, accelerates cardiac fibrosis, and increases heart failure. Mechanistically, CYLD interacts with plakoglobin and removes lysine 63-linked polyubiquitin chains from plakoglobin. The deubiquitination of plakoglobin enhances its interaction with the desmoplakin/end-binding protein 1 complex localized at the microtubule plus end, thereby promoting microtubule-dependent transport of connexin 43 (Cx43), a key component of gap junctions, to the cell membrane. These findings establish CYLD as a critical player in regulating gap junction assembly and have important implications in heart development and diseases.

Targeting the HDAC6-Cilium Axis Ameliorates the Pathological Changes Associated with Retinopathy of Prematurity.

Retinopathy of prematurity (ROP) is one of the leading causes of childhood visual impairment and blindness. However, there are still very few effective pharmacological interventions for ROP. Histone deacetylase 6 (HDAC6)-mediated disassembly of photoreceptor cilia has recently been implicated as an early event in the pathogenesis of ROP. Herein it is shown that enhanced expression of HDAC6 by intravitreal injection of adenoviruses encoding HDAC6 induces the typical pathological changes associated with ROP in mice, including disruption of the membranous disks of photoreceptor outer segments and a decrease in electroretinographic amplitudes. Hdac6 transgenic mice exhibit similar ROP-related defects in retinal structures and functions and disassembly of photoreceptor cilia, whereas Hdac6 knockout mice are resistant to oxygen change-induced retinal defects. It is further shown that blocking HDAC6-mediated cilium disassembly by intravitreal injection of small-molecule compounds protect mice from ROP-associated retinal defects. The findings indicate that pharmacological targeting of the HDAC6-cilium axis may represent a promising strategy for the prevention of ROP.

ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis.

Despite the importance of cilia in cell signaling and motility, the molecular mechanisms regulating cilium formation remain incompletely understood. Herein, we characterize enkurin domain-containing protein 1 (ENKD1) as a novel centrosomal protein that mediates the removal of centriolar coiled-coil protein 110 (CP110) from the mother centriole to promote ciliogenesis. We show that Enkd1 knockout mice possess ciliogenesis defects in multiple organs. Super-resolution microscopy reveals that ENKD1 is a stable component of the centrosome throughout the ciliogenesis process. Simultaneous knockdown of ENKD1 and CP110 significantly reverses the ciliogenesis defects induced by ENKD1 depletion. Protein interaction analysis shows that ENKD1 competes with centrosomal protein 97 (CEP97) in binding to CP110. Depletion of ENKD1 enhances the CP110-CEP97 interaction and detains CP110 at the mother centriole. These findings thus identify ENKD1 as a centrosomal protein and uncover a novel mechanism controlling CP110 removal from the mother centriole for the initiation of ciliogenesis.

ENKD1 promotes epidermal stratification by regulating spindle orientation in basal keratinocytes.

Stratification of the epidermis is essential for the barrier function of the skin. However, the molecular mechanisms governing epidermal stratification are not fully understood. Herein, we demonstrate that enkurin domain-containing protein 1 (ENKD1) contributes to epidermal stratification by modulating the cell-division orientation of basal keratinocytes. The epidermis of Enkd1 knockout mice is thinner than that of wild-type mice due to reduced generation of suprabasal cells from basal keratinocytes through asymmetric division. Depletion of ENKD1 impairs proper orientation of the mitotic spindle and delays mitotic progression in cultured cells. Mechanistic investigation further reveals that ENKD1 is a novel microtubule-binding protein that promotes the stability of astral microtubules. Introduction of the microtubule-binding domain of ENKD1 can largely rescue the spindle orientation defects in ENKD1-depleted cells. These findings establish ENKD1 as a critical regulator of astral microtubule stability and spindle orientation that stimulates epidermal stratification in mammalian cells.

The specialized mitotic behavior of human embryonic stem cells.

Human embryonic stem cells (hESCs) are self-renewing and pluripotent cells that originate from the inner cell mass of the blastocyst. Mitosis is fundamental to organism survival and reproduction and is responsible for the equal distribution of duplicated chromosomes into daughter cells. Mitotic dysfunction is associated with a wide variety of human diseases, not least cancer. hESCs have a unique cell cycle distribution, but it is unclear exactly how the mitotic activity of hESCs is related to their proliferation and differentiation. Here, we established a cell line of hESCs stably expressing GFP-α-tubulin and mCherry-H2B by lentiviral infection to analyze and visualize mitosis in detail. During metaphase, the mitotic spindle was smaller and wider and contained a greater proportion of astral microtubules than normal cells. In addition, spindle microtubules were more stable, and chromosome alignment was faster in hESCs than in somatic cells. We also found that the spindle assembly checkpoint was functional in hESCs. These findings thus reveal a specialized mitotic behavior of hESCs.

Functions and Diseases of the Retinal Pigment Epithelium.

The retinal pigment epithelium is a fundamental component of the retina that plays essential roles in visual functions. Damage to the structure and function of the retinal pigment epithelium leads to a variety of retinopathies, and there is currently no curative therapy for these disorders. Therefore, studying the relationship between the development, function, and pathobiology of the retinal pigment epithelium is important for the prevention and treatment of retinopathies. Here we review the function of the retinal pigment epithelium and its relevance to the pathobiology, and discuss potential strategies for the treatment of retinopathies. In doing so, we provide new viewpoints outlining new ideas for the future study and treatment of retinopathies.

CYLD deficiency causes auditory neuropathy due to reduced neurite outgrowth.

BACKGROUND: Auditory neuropathy is a cause of hearing loss that has been studied in a number of animal models. Signal transmission from hair cells to spiral ganglion neurons plays an important role in normal hearing. CYLD is a microtubule-binding protein, and deubiquitinase involved in the regulation of various cellular processes. In this study, we used Cyld knockout (KO) mice and nerve cell lines to examine whether CYLD is associated with auditory neuropathy. METHODS: Hearing of Cyld KO mice was studied using the TDT RZ6 auditory physiology workstation. The expression and localization of CYLD in mouse cochlea and cell lines were examined by RT-PCR, immunoblotting, and immunofluorescence. CYLD expression was knocked down in SH-SY5Y cells by shRNAs and in PC12 and N2A cells by siRNAs. Nerve growth factor and retinoic acid were used to induce neurite outgrowth, and the occurrence and length of neurites were statistically analyzed between knockdown and control groups. RESULTS: Cyld KO mice had mild hearing impairment. Moreover, CYLD was widely expressed in mouse cochlear tissues and different nerve cell lines. Knocking down CYLD significantly reduced the length and proportion of neurites growing from nerve cells. CONCLUSIONS: The abnormal hearing of Cyld KO mice might be caused by a decrease in the length and number of neurites growing from auditory nerve cells in the cochlea, suggesting that CYLD is a key protein affecting hearing.

Potential role for the tumor suppressor CYLD in brain and notochord development.

BACKGROUND: The cylindromatosis (CYLD) tumor suppressor is a microtubule-associated deubiquitinase that plays a critical role in the regulation of cell signaling and contributes to a variety of physiological and pathological processes. However, the functions of CYLD in zebrafish are less well known, particularly with regard to their development and physiology. In this context, we investigated the loss of function of CYLD in zebrafish via transcription activator-like effector nuclease (TALEN)-based gene deletion. METHODS: Semi-quantitative RT-PCR was used to quantify CYLD mRNA expression in zebrafish embryos at various developmental stages. We also performed whole-mount in situ hybridization to further assess the dynamic expression and distribution of CYLD in the entire zebrafish embryos at different stages. In addition, we deleted CYLD in zebrafish with TALENs to investigate its potential impact on embryonic development. RESULTS: The expression of CYLD mRNA varied during early embryonic development. The CYLD mRNA localized to the brain and notochord of developing zebrafish embryos. Homozygous deletion of CYLD resulted in embryonic death before 8 h post-fertilization. CONCLUSIONS: CYLD appears to play an important role in central nervous system development in zebrafish. Although severe embryonic death restricted analysis of homozygous mutants, further research into the role of CYLD in central nervous system development is warranted.

HIV-1 exposure promotes PKG1-mediated phosphorylation and degradation of stathmin to increase epithelial barrier permeability.

Exposure of mucosal epithelial cells to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is known to disrupt epithelial cell junctions by impairing stathmin-mediated microtubule depolymerization. However, the pathological significance of this process and its underlying molecular mechanism remain unclear. Here we show that treatment of epithelial cells with pseudotyped HIV-1 viral particles or recombinant gp120 protein results in the activation of protein kinase G 1 (PKG1). Examination of epithelial cells by immunofluorescence microscopy reveals that PKG1 activation mediates the epithelial barrier damage upon HIV-1 exposure. Immunoprecipitation experiments show that PKG1 interacts with stathmin and phosphorylates stathmin at serine 63 in the presence of gp120. Immunoprecipitation and immunofluorescence microscopy further demonstrate that PKG1-mediated phosphorylation of stathmin promotes its autophagic degradation by enhancing the interaction between stathmin and the autophagy adaptor protein p62. Collectively, these results suggest that HIV-1 exposure exploits the PKG1/stathmin axis to affect the microtubule cytoskeleton and thereby perturbs epithelial cell junctions. Our findings reveal a novel molecular mechanism by which exposure to HIV-1 increases epithelial permeability, which has implications for the development of effective strategies to prevent mucosal HIV-1 transmission.