Keratin-Based Nanoparticles with Tumor-Targeting and Cascade Catalytic Capabilities for the Combinational Oxidation Phototherapy of Breast Cancer.
Lu Ting-Yu,Lu Wei-Fan,Wang Yin-Hsu,Liao Mei-Yi,Wei Yang,Fan Yu-Jui,Chuang Er-Yuan,Yu Jiashing
ACS applied materials & interfaces
Photodynamic therapy (PDT) holds tantalizing prospects of a prominent cancer treatment strategy. However, its efficacy remains limited by virtue of the hypoxic tumor microenvironment and the inadequate tumor-targeted delivery of photosensitizers, and these can be further exacerbated by the lack of development of a well-controlled nitric oxide (NO) release system at the target site. Inspired by Chinese medicine, we propose a revealing new keratin application. Keratin has garnered attention as an NO generator; however, its oncological use has rarely been investigated. We hypothesized that the incorporation of a phenylboronic acid (PBA) targeting ligand/methylene blue (MB) photosensitizer with a keratin NO donor would facilitate precise tumor delivery, enhancing PDT. Herein, we demonstrated that MB@keratin/PBA/d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) nanoparticles (MB@KPTNPs) specifically targeted breast cancer cells and effectively suppressed their growth. Through MB-mediated biometabolism, the endocytic MB@KPTNPs produced a sufficient amount of intracellular NO that reduced the glutathione level while boosting the efficiency of PDT. A therapeutic combination of NO/PDT was therefore achieved, resulting in significant inhibition of both tumor growth and lung metastasis. These findings underscore the importance of utilizing keratin-based nanoparticles that simultaneously combine targeting of the tumor and self-generating NO with a cascading catalytic ability as a novel oxidation therapeutic strategy for enhancing PDT.
10.1021/acsami.1c10160
Keratin 86 is up-regulated in the uterus during implantation, induced by oestradiol.
Zhang He,Zhao Huashan,Wang Xi,Cui Xiaolin,Jin Lingling
BMC developmental biology
BACKGROUND:Uterine receptivity is one of the determinants of embryo implantation, which is responsible for pregnancy success. Aberrant embryo implantation due to disrupted uterine receptivity is usually found in ovarian hyperstimulation induced hyperoestrogen patients. RESULTS:This study identified keratin 86 (KRT86), a fibrous structural protein, which was upregulated in uterine endometrium during peri-implantation. Using a hyperoestrogen mouse model established in a previous study, we found abnormal oestradiol (E2) levels during pre-implantation could trigger high expression of Krt86 in the uterine epithelium. In an ovariectomised mouse model, combining oestrogen receptors ERα and ERβ knockout mice models, uterine Krt86 was found to be up-regulated after E2 treatment, mediated by nuclear ERα. Furthermore, we found progesterone (P4) could ameliorate Krt86 expression, induced by abnormal E2. CONCLUSIONS:These results revealed the dynamic expression and regulation of Krt86, especially in hyperoestrogen treated mice, indicating it might act as a marker for non-receptive uterus.
10.1186/s12861-020-0208-6
Keratin 6 regulates collective keratinocyte migration by altering cell-cell and cell-matrix adhesion.
Wang Fengrong,Chen Song,Liu Hans B,Parent Carole A,Coulombe Pierre A
The Journal of cell biology
The a and b isoforms of keratin 6 (K6), a type II intermediate filament (IF) protein, are robustly induced upon injury to interfollicular epidermis. We previously showed that complete loss of K6a/K6b stimulates keratinocyte migration, correlating with enhanced Src activity. In this study, we demonstrate that this property is cell autonomous, depends on the ECM, and results from elevated speed, enhanced directionality, and an increased rate of focal adhesion disassembly. We show that myosin IIA interacts with K6a/K6b, that its levels are markedly reduced in -null keratinocytes, and that inhibiting myosin ATPase activity normalizes the enhanced migration potential of -null cells. Desmoplakin, which mediates attachment of IFs to desmosomes, is also expressed at reduced levels and is mislocalized to the nucleus in -null cells, correlating with defects in cell adhesion. These findings reveal that K6a/K6b modulate keratinocyte migration by regulating cell-matrix and cell-cell adhesion and highlight a role for keratins in collective cell migration.
10.1083/jcb.201712130
Keratin 79 is a PPARA target that is highly expressed by liver damage.
Biochemical and biophysical research communications
Keratins are key structural proteins found in skin and other epithelial tissues. Keratins also protect epithelial cells from damage or stress. Fifty-four human keratins were identified and classified into two families, type I and type II. Accumulating studies showed that keratin expression is highly tissue-specific and used as a diagnostic marker for human diseases. Notably, keratin 79 (KRT79) is type II cytokeratin that was identified as regulator of hair canal morphogenesis and regeneration in skin, but its role in liver remains unclear. KRT79 is undetectable in normal mouse but its expression is significantly increased by the PPARA agonist WY-14643 and fenofibrate, and completely abolished in Ppara-null mice. The Krt79 gene has functional PPARA binding element between exon 1 and exon 2. Hepatic Krt79 is regulated by HNF4A and HER2. Moreover, hepatic KRT79 is also significantly elevated by fasting- and high-fat diet-induced stress, and these increases are completely abolished in Ppara-null mice. These findings suggest that hepatic KRT79 is controlled by PPARA and is highly associated with liver damage. Thus, KRT79 may be considered as a diagnostic marker for human liver diseases.
10.1016/j.bbrc.2023.01.071
Activation of the C3a anaphylatoxin receptor inhibits keratinocyte proliferation by regulating keratin 6, keratin 16, and keratin 17 in psoriasis.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Emerging evidence suggests that signaling through the C3a anaphylatoxin receptor (C3aR) protects against various inflammation-related diseases. However, the role of C3aR in psoriasis remains unknown. The purpose of this study was to investigate the possible protective role of C3aR in psoriasis and to explore the underlying molecular mechanisms. We initially found that the psoriatic epidermis exhibited significantly decreased C3aR expression. C3aR showed protective roles in mouse models of imiquimod (IMQ)- and interleukin-23-induced psoriasis. Furthermore, increased epidermal thickness and keratin 6 (K6), K16, and K17 expression occurred in the ears and backs of C3aR mice. Pharmacological treatment with a C3aR agonist ameliorated IMQ-induced psoriasiform lesions in mice and decreased the expression of K6, K16, and K17. Additionally, the signal transducer and activator of transcription 3 (STAT3) pathway participated in the protective function of C3aR. More importantly, the expression levels of K6, K16, and K17 in keratinocytes were all restored in HaCaT cells transfected with a C3aR-overexpression plasmid after treating them with colivelin (a STAT3 activator). Our findings demonstrate that C3aR protects against the development of psoriasis and suggest that C3aR confers protection by negatively regulating K6, K16, and K17 expression in a STAT3-dependent manner, thus inhibiting keratinocyte proliferation and helping reverse the pathogenesis of psoriasis.
10.1096/fj.202101458R
A role for keratin 17 during DNA damage response and tumor initiation.
Proceedings of the National Academy of Sciences of the United States of America
High levels of the intermediate filament protein keratin 17 (K17) are associated with poor prognoses for several human carcinomas. Studies in mouse models have shown that K17 expression is positively associated with growth, survival, and inflammation in skin and that lack of K17 delays onset of tumorigenesis. K17 occurs in the nucleus of human and mouse tumor keratinocytes where it impacts chromatin architecture, gene expression, and cell proliferation. We report here that K17 is induced following DNA damage and promotes keratinocyte survival. The presence of nuclear K17 is required at an early stage of the double-stranded break (DSB) arm of the DNA damage and repair (DDR) cascade, consistent with its ability to associate with key DDR effectors, including γ-H2A.X, 53BP1, and DNA-PKcs. Mice lacking K17 or with attenuated K17 nuclear import showed curtailed initiation in a two-step skin carcinogenesis paradigm. The impact of nuclear-localized K17 on DDR and cell survival provides a basis for the link between K17 induction and poor clinical outcomes for several human carcinomas.
10.1073/pnas.2020150118
PP2 protects from keratin mutation-associated liver injury and filament disruption via SRC kinase inhibition in male but not female mice.
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Hepatocyte keratin polypeptides 8/18 (K8/K18) are unique among intermediate filaments proteins (IFs) in that their mutation predisposes to, rather than causes, human disease. Mice that overexpress human K18 R90C manifest disrupted hepatocyte keratin filaments with hyperphosphorylated keratins and predisposition to Fas-induced liver injury. We hypothesized that high-throughput screening will identify compounds that protect the liver from mutation-triggered predisposition to injury. APPROACH AND RESULTS:Using A549 cells transduced with a lentivirus K18 construct and high-throughput screening, we identified the SRC-family tyrosine kinases inhibitor, PP2, as a compound that reverses keratin filament disruption and protects from apoptotic cell death caused by K18 R90C mutation at this highly conserved arginine. PP2 also ameliorated Fas-induced apoptosis and liver injury in male but not female K18 R90C mice. The PP2 male selectivity is due to its lower turnover in male versus female livers. Knockdown of SRC but not another kinase target of PP2, protein tyrosine kinase 6, in A549 cells abrogated the hepatoprotective effect of PP2. Phosphoproteomic analysis and validation showed that the protective effect of PP2 associates with Ser/Thr but not Tyr keratin hypophosphorylation, and differs from the sex-independent effect of the Ser/Thr kinase inhibitor PKC412. Inhibition of RAF kinase, a downstream target of SRC, by vemurafenib had a similar protective effect to PP2 in A549 cells and male K18 R90C mice. CONCLUSIONS:PP2 protects, in a male-selective manner, keratin mutation-induced mouse liver injury by inhibiting SRC-triggered downstream Ser/Thr phosphorylation of K8/K18, which is phenocopied by RAF kinase inhibitor vemurafenib. The PP2/vemurafenib-associated findings, and their unique mechanisms of action, further support the potential role of select kinase inhibition as therapeutic opportunities for keratin and other IF-associated human diseases.
10.1002/hep.32574
Decreased levels of keratin 8 sensitize mice to streptozotocin-induced diabetes.
Alam C M,Silvander J S G,Helenius T O,Toivola D M
Acta physiologica (Oxford, England)
AIM:Diabetes is a result of an interplay between genetic, environmental and lifestyle factors. Keratin intermediate filaments are stress proteins in epithelial cells, and keratin mutations predispose to several human diseases. However, the involvement of keratins in diabetes is not well known. K8 and its partner K18 are the main β-cell keratins, and knockout of K8 (K8 ) in mice causes mislocalization of glucose transporter 2, mitochondrial defects, reduced insulin content and altered systemic glucose/insulin control. We hypothesize that K8/K18 offer protection during β-cell stress and that decreased K8 levels contribute to diabetes susceptibility. METHODS:K8-heterozygous knockout (K8 ) and wild-type (K8 ) mice were used to evaluate the influence of keratin levels on endocrine pancreatic function and diabetes development under basal conditions and after T1D streptozotocin (STZ)-induced β-cell stress and T2D high-fat diet (HFD). RESULTS:Murine K8 endocrine islets express ~50% less K8/K18 compared with K8 . The decreased keratin levels have little impact on basal systemic glucose/insulin regulation, β-cell health or insulin levels. Diabetes incidence and blood glucose levels are significantly higher in K8 mice after low-dose/chronic STZ treatment, and STZ causes more β-cell damage and polyuria in K8 compared with K8 . K8 appears upregulated 5 weeks after STZ treatment in K8 islets but not in K8 . K8 mice showed no major susceptibility risk to HFD compared to K8 . CONCLUSION:Partial K8 deficiency reduces β-cell stress tolerance and aggravates diabetes development in response to STZ, while there is no major susceptibility to HFD.
10.1111/apha.13085
Stress Keratin 17 Expression in Head and Neck Cancer Contributes to Immune Evasion and Resistance to Immune-Checkpoint Blockade.
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:We investigated whether in human head and neck squamous cell carcinoma (HNSCC) high levels of expression of stress keratin 17 (K17) are associated with poor survival and resistance to immunotherapy. EXPERIMENTAL DESIGN:We investigated the role of K17 in regulating both the tumor microenvironment and immune responsiveness of HNSCC using a syngeneic mouse HNSCC model, MOC2. MOC2 gives rise to immunologically cold tumors that are resistant to immune-checkpoint blockade (ICB). We engineered multiple, independent K17 knockout (KO) MOC2 cell lines and monitored their growth and response to ICB. We also measured K17 expression in human HNSCC of patients undergoing ICB. RESULTS:MOC2 tumors were found to express K17 at high levels. When knocked out for K17 (K17KO MOC2), these cells formed tumors that grew slowly or spontaneously regressed and had a high CD8+ T-cell infiltrate in immunocompetent syngeneic C57BL/6 mice compared with parental MOC2 tumors. This phenotype was reversed when we depleted mice for T cells. Whereas parental MOC2 tumors were resistant to ICB treatment, K17KO MOC2 tumors that did not spontaneously regress were eliminated upon ICB treatment. In a cohort of patients with HNSCC receiving pembrolizumab, high K17 expression correlated with poor response. Single-cell RNA-sequencing analysis revealed broad differences in the immune landscape of K17KO MOC2 tumors compared with parental MOC2 tumors, including differences in multiple lymphoid and myeloid cell types. CONCLUSIONS:We demonstrate that K17 expression in HNSCC contributes to immune evasion and resistance to ICB treatment by broadly altering immune landscapes of tumors.
10.1158/1078-0432.CCR-21-3039
Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells.
JCI insight
Keratin expression dynamically changes in airway basal cells (BCs) after acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In bronchiolitis obliterans (BO) after lung transplantation, BC clonogenicity declines, which is associated with a switch from keratin15 (Krt15) to keratin14 (Krt14). We investigated these keratins' roles using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs did not differentiate into club and ciliated cells but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo after injury. Krt14, but not Krt15, bound the tumor suppressor stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, whereas dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts with Krt14-KO phenotype and resembles the phenotype in BO with decreased clonogenicity, increased Krt14, and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior, which is relevant in chronic disease states like BO.
10.1172/jci.insight.162041
Desmoplakin Maintains Transcellular Keratin Scaffolding and Protects From Intestinal Injury.
Gross Annika,Zhou Biaohuan,Bewersdorf Lisa,Schwarz Nicole,Schacht Gabriel M,Boor Peter,Hoeft Konrad,Hoffmann Bernd,Fuchs Elaine,Kramann Rafael,Merkel Rudolf,Leube Rudolf E,Strnad Pavel
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell-cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome-keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia. METHODS:We analyzed the intestine-specific villin-Cre DSP (DSP) and the combined intestine-specific DSG2/DSP (ΔDsg2/Dsp) knockout mice. Cross-breeding with keratin 8-yellow fluorescent protein knock-in mice and generation of organoids was performed to visualize the keratin network. A Dsp-deficient colorectal carcinoma HT29-derived cell line was generated and the role of Dsp in adhesion and mechanical stress was studied in dispase assays, after exposure to uniaxial cell stretching and during scratch assay. RESULTS:The intestine of DSP mice was histopathologically inconspicuous. Intestinal epithelial cells, however, showed an accelerated migration along the crypt and an enhanced shedding into the lumen. Increased intestinal permeability and altered levels of desmosomal proteins were detected. An inconspicuous phenotype also was seen in ΔDsg2/Dsp mice. After dextran sodium sulfate treatment, DSP mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSP/keratin 8-yellow fluorescent protein mice and organoids derived from these mice presented a collapsed keratin network. The level, phosphorylation status, and solubility of keratins were not affected. Dsp-deficient HT29 cells had an impaired cell adhesion and suffered from increased cellular damage after stretch. CONCLUSIONS:Our results show that Dsp is required for proper keratin network architecture in intestinal epithelia, mechanical resilience, and adhesion, thereby protecting from injury.
10.1016/j.jcmgh.2021.12.009
Keratin-mediated hair growth and its underlying biological mechanism.
Communications biology
Here we show that intradermal injection of keratin promotes hair growth in mice, which results from extracellular interaction of keratin with hair forming cells. Extracellular application of keratin induces condensation of dermal papilla cells and the generation of a P-cadherin-expressing cell population (hair germ) from outer root sheath cells via keratin-mediated microenvironmental changes. Exogenous keratin-mediated hair growth is reflected by the finding that keratin exposure from transforming growth factor beta 2 (TGFβ2)-induced apoptotic outer root sheath cells appears to be critical for dermal papilla cell condensation and P-cadherin-expressing hair germ formation. Immunodepletion or downregulation of keratin released from or expressed in TGFβ2-induced apoptotic outer root sheath cells negatively influences dermal papilla cell condensation and hair germ formation. Our pilot study provides an evidence on initiating hair regeneration and insight into the biological function of keratin exposed from apoptotic epithelial cells in tissue regeneration and development.
10.1038/s42003-022-04232-9
Simultaneous control of infection and inflammation with keratin-derived antibacterial peptides targeting TLRs and co-receptors.
Science translational medicine
Controlling infection-driven inflammation is a major clinical dilemma because of limited therapeutic options and possible adverse effects on microbial clearance. Compounding this difficulty is the continued emergence of drug-resistant bacteria, where experimental strategies aiming to augment inflammatory responses for enhanced microbial killing are not applicable treatment options for infections of vulnerable organs. As with corneal infections, severe or prolonged inflammation jeopardizes corneal transparency, leading to devastating vision loss. We hypothesized that keratin 6a-derived antimicrobial peptides (KAMPs) may be a two-pronged remedy capable of tackling bacterial infection and inflammation at once. We used murine peritoneal neutrophils and macrophages, together with an in vivo model of sterile corneal inflammation, to find that nontoxic and prohealing KAMPs with natural 10- and 18-amino acid sequences suppressed lipoteichoic acid (LTA)- and lipopolysaccharide (LPS)-induced NFκB and IRF3 activation, proinflammatory cytokine production, and phagocyte recruitment independently of their bactericidal function. Mechanistically, KAMPs not only competed with bacterial ligands for cell surface Toll-like receptor (TLR) and co-receptors (MD2, CD14, and TLR2) but also reduced cell surface availability of TLR2 and TLR4 through promotion of receptor endocytosis. Topical KAMP treatment effectively alleviated experimental bacterial keratitis, as evidenced by substantial reductions of corneal opacification, inflammatory cell infiltration, and bacterial burden. These findings reveal the TLR-targeting activities of KAMPs and demonstrate their therapeutic potential as a multifunctional drug for managing infectious inflammatory disease.
10.1126/scitranslmed.ade2909
Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.
Jang Kwi-Hoon,Yoon Han-Na,Lee Jongeun,Yi Hayan,Park Sang-Yoon,Lee So-Young,Lim Younglan,Lee Hyoung-Joo,Cho Jin-Won,Paik Young-Ki,Hancock Williams S,Ku Nam-On
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Keratin 8 (K8) and keratin 18 (K18) are the intermediate filament proteins whose phosphorylation/transamidation associate with their aggregation in Mallory-Denk bodies found in patients with various liver diseases. However, the functions of other post-translational modifications in keratins related to liver diseases have not been fully elucidated. Here, using a site-specific mutation assay combined with nano-liquid chromatography-tandem mass spectrometry, we identified K8-Lys108 and K18-Lys187/426 as acetylation sites, and K8-Arg47 and K18-Arg55 as methylation sites. Keratin mutation (Arg-to-Lys/Ala) at the methylation sites, but not the acetylation sites, led to decreased stability of the keratin protein. We compared keratin acetylation/methylation in liver disease-associated keratin variants. The acetylation of K8 variants increased or decreased to various extents, whereas the methylation of K18-del65-72 and K18-I150V variants increased. Notably, the highly acetylated/methylated K18-I150V variant was less soluble and exhibited unusually prolonged protein stability, which suggests that additional acetylation of highly methylated keratins has a synergistic effect on prolonged stability. Therefore, the different levels of acetylation/methylation of the liver disease-associated variants regulate keratin protein stability. These findings extend our understanding of how disease-associated mutations in keratins modulate keratin acetylation and methylation, which may contribute to disease pathogenesis.-Jang, K.-H., Yoon, H.-N., Lee, J., Yi, H., Park, S.-Y., Lee, S.-Y., Lim, Y., Lee, H.-J., Cho, J.-W., Paik, Y.-K., Hancock, W. S., Ku, N.-O. Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.
10.1096/fj.201800263RR
SIRT6 pharmacological inhibition delays skin cancer progression in the squamous cell carcinoma.
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Sirtuin 6 (SIRT6) has a critical role in cutaneous Squamous Cell Carcinoma (cSCC): SIRT6 silencing in skin SCC cells has pro-differentiating effects and SIRT6 deletion abrogated DMBA-TPA-induced skin tumorigenesis in mice. On the other hand, SIRT6 acts as tumor suppressor in SCC by enhancing glycolysis in tumor propagating cells. Herein, pharmacological modulation of SIRT6 deacetylase activity was investigated in cSCC, with S6 (inhibitor) or MDL-800 (activator). In cSCC cells, S6 recreated the pro-differentiating effects of SIRT6 silencing, as the levels of Keratin 1, Keratin 10 and Loricrin were upregulated compared to controls. Next, the effects of SIRT6 pharmacological modulation were evaluated in a DMBA-TPA-induced skin cancer mouse model. Mice treated with the inhibitor S6 in a preventive approach, i.e. at the beginning of the promotion stage, presented reduced number and size of papillomas, compared to the controls. The epidermal hyperproliferation marker Keratin 6 and the cSCC marker Keratin 8 were less abundant when SIRT6 was inhibited. In S6-treated lesions, the Epithelial-Mesenchymal Transition (EMT) markers Zeb1 and Vimentin were less expressed compared to untreated lesions. In a therapeutic approach, i.e. treatment starting after papilloma appearance, the S6 group presented reduced papillomas (number and size), whereas MDL-800-treated mice displayed an opposite trend. In S6-treated lesions, Keratin 6 and Keratin 8 were less expressed, EMT was less advanced, with a higher E-cadherin/Vimentin ratio, indicating a delayed carcinogenesis when SIRT6 was inhibited. Our results confirm that SIRT6 plays a role in skin carcinogenesis and suggest SIRT6 pharmacological inhibition as a promising strategy in cSCC.
10.1016/j.biopha.2023.115326