Translational profiling identifies sex-specific metabolic and epigenetic reprogramming of cortical microglia/macrophages in APPPS1-21 mice with an antibiotic-perturbed-microbiome.

BACKGROUND: Microglia, the brain-resident macrophages perform immune surveillance and engage with pathological processes resulting in phenotype changes necessary for maintaining homeostasis. In preceding studies, we showed that antibiotic-induced perturbations of the gut microbiome of APPPS1-21 mice resulted in significant attenuation in Aß amyloidosis and altered microglial phenotypes that are specific to male mice. The molecular events underlying microglial phenotypic transitions remain unclear. Here, by generating 'APPPS1-21-CD11br' reporter mice, we investigated the translational state of microglial/macrophage ribosomes during their phenotypic transition and in a sex-specific manner. METHODS: Six groups of mice that included WT-CD11br, antibiotic (ABX) or vehicle-treated APPPS1-21-CD11br males and females were sacrificed at 7-weeks of age (n = 15/group) and used for immunoprecipitation of microglial/macrophage polysomes from cortical homogenates using anti-FLAG antibody. Liquid chromatography coupled to tandem mass spectrometry and label-free quantification was used to identify newly synthesized peptides isolated from polysomes. RESULTS: We show that ABX-treatment leads to decreased Aß levels in male APPPS1-21-CD11br mice with no significant changes in females. We identified microglial/macrophage polypeptides involved in mitochondrial dysfunction and altered calcium signaling that are associated with Aß-induced oxidative stress. Notably, female mice also showed downregulation of newly-synthesized ribosomal proteins. Furthermore, male mice showed an increase in newly-synthesized polypeptides involved in FcγR-mediated phagocytosis, while females showed an increase in newly-synthesized polypeptides responsible for actin organization associated with microglial activation. Next, we show that ABX-treatment resulted in substantial remodeling of the epigenetic landscape, leading to a metabolic shift that accommodates the increased bioenergetic and biosynthetic demands associated with microglial polarization in a sex-specific manner. While microglia in ABX-treated male mice exhibited a metabolic shift towards a neuroprotective phenotype that promotes Aß clearance, microglia in ABX-treated female mice exhibited loss of energy homeostasis due to persistent mitochondrial dysfunction and impaired lysosomal clearance that was associated with inflammatory phenotypes. CONCLUSIONS: Our studies provide the first snapshot of the translational state of microglial/macrophage cells in a mouse model of Aß amyloidosis that was subject to ABX treatment. ABX-mediated changes resulted in metabolic reprogramming of microglial phenotypes to modulate immune responses and amyloid clearance in a sex-specific manner. This microglial plasticity to support neuro-energetic homeostasis for its function based on sex paves the path for therapeutic modulation of immunometabolism for neurodegeneration.

An APP ectodomain mutation outside of the Aß domain promotes Aß production in vitro and deposition in vivo.

Familial Alzheimer's disease (FAD)-linked mutations in the APP gene occur either within the Aß-coding region or immediately proximal and are located in exons 16 and 17, which encode Aß peptides. We have identified an extremely rare, partially penetrant, single nucleotide variant (SNV), rs145081708, in APP that corresponds to a Ser198Pro substitution in exon 5. We now report that in stably transfected cells, expression of APP harboring the S198P mutation (APPS198P) leads to elevated production of Aß peptides by an unconventional mechanism in which the folding and exit of APPS198P from the endoplasmic reticulum is accelerated. More importantly, coexpression of APP S198P and the FAD-linked PS1ΔE9 variant in the brains of male and female transgenic mice leads to elevated steady-state Aß peptide levels and acceleration of Aß deposition compared with age- and gender-matched mice expressing APP and PS1ΔE9. This is the first AD-linked mutation in APP present outside of exons 16 and 17 that enhances Aß production and deposition.

Negative evidence for a role of APH1B T27I variant in Alzheimer's disease.

γ-secretase is a macromolecular complex that catalyzes intramembranous hydrolysis of more than 100 membrane-bound substrates. The complex is composed of presenilin (PS1 or PS2), anterior pharynx defect-1 (APH-1), nicastrin (NCT) and PEN-2 and early-onset; autosomal dominant forms of Alzheimer's disease (AD) are caused by inheritance of mutations of PS. No mutations in genes encoding NCT, or PEN-2 have been identified to date that cause AD. In this regard, a large genetic meta-analysis of four cohorts consisting of more than 600 000 individuals identified a common missense variant, rs117618017 in the APH1B gene that results in a T27I mutation, as a novel genome-wide significant locus. In order to confirm the findings that rs117618017 is associated with risk of AD, we performed a genetic screen from deep whole genome sequencing of the large NIMH family-based Alzheimer's Disease (AD) dataset. In parallel, we sought to uncover potential molecular mechanism(s) by which APH-1B T27I might be associated with AD by generating stable HEK293 cell lines, wherein endogenous APH-1A and APH-1B expression was silenced and into which either the wild type APH-1B or the APH-1B T27I variant was stably expressed. We then tested the impact of expressing either the wild type APH-1B or the APH-1B T27I variant on γ-secretase processing of human APP, the murine Notch derivative mNΔE and human neuregulin-1. We now report that we fail to confirm the association of rs1047552 with AD in our cohort and that cells expressing the APH-1B T27I variant show no discernable impact on the γ-secretase processing of established substrates compared with cells expressing wild-type APH-1B.

SIRT6 promotes COX-2 expression and acts as an oncogene in skin cancer.

SIRT6 is a SIR2 family member that regulates multiple molecular pathways involved in metabolism, genomic stability, and aging. It has been proposed previously that SIRT6 is a tumor suppressor in cancer. Here, we challenge this concept by presenting evidence that skin-specific deletion of SIRT6 in the mouse inhibits skin tumorigenesis. SIRT6 promoted expression of COX-2 by repressing AMPK signaling, thereby increasing cell proliferation and survival in the skin epidermis. SIRT6 expression in skin keratinocytes was increased by exposure to UVB light through activation of the AKT pathway. Clinically, we found that SIRT6 was upregulated in human skin squamous cell carcinoma. Taken together, our results provide evidence that SIRT6 functions as an oncogene in the epidermis and suggest greater complexity to its role in epithelial carcinogenesis.

Deregulation of XPC and CypA by cyclosporin A: an immunosuppression-independent mechanism of skin carcinogenesis.

Skin cancer is the most common malignancy in organ transplant recipients, causing serious morbidity and mortality. Preventing and treating skin cancer in these individuals has been extraordinarily challenging. Following organ transplantation, cyclosporin A (CsA) has been used as an effective immunosuppressive to prevent rejection. Therefore immunosuppression has been widely assumed to be the major cause for increased skin carcinogenesis. However, the mechanism of skin carcinogenesis in organ transplant recipients has not been understood to date; specifically, it remains unknown whether these cancers are immunosuppression dependent or independent. Here, using both immunocompromised nude mice which are defective in mature T lymphocytes as an in vivo model and human keratinocytes as an in vitro model, we showed that CsA impairs genomic integrity in the response of keratinocytes to ultra violet B (UVB). Following UVB radiation, CsA inhibited UVB-induced DNA damage repair by suppressing the transcription of the DNA repair factor xeroderma pigmentosum C (XPC). In addition, CsA compromised the UVB-induced checkpoint function by upregulating the molecular chaperone protein cyclophilin A (CypA). XPC mRNA levels were lower, whereas CypA mRNA and protein levels were higher in human skin cancers than in normal skin. CsA-induced phosphoinositide 3-kinase(PI3K)/AKT activation was required for both XPC suppression and CypA upregulation. Blocking UVB damage or inhibiting the PI3K/AKT pathway prevented CsA-sensitized skin tumorigenesis. Our findings identified deregulation of XPC and CypA as key targets of CsA, and UVB damage and PI3K/AKT activation as two principal drivers for CsA-sensitized skin tumorigenesis, further supporting an immunosuppression-independent mechanism of CsA action on skin tumorigenesis.

Nrf1 CNC-bZIP protein promotes cell survival and nucleotide excision repair through maintaining glutathione homeostasis.

Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.

PTEN positively regulates UVB-induced DNA damage repair.

Nonmelanoma skin cancer is the most common cancer in the United States, where DNA-damaging ultraviolet B (UVB) radiation from the sun remains the major environmental risk factor. However, the critical genetic targets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is a predisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cell carcinoma (SCC), levels of PTEN were reduced compared with skin lacking these lesions. Likewise, there was a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCCs, supporting a key role for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity of global genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolonged UVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels in PTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essential genomic gatekeeper in the skin through its ability to positively regulate XPC-dependent GG-NER following DNA damage.

Caffeine promotes ultraviolet B-induced apoptosis in human keratinocytes without complete DNA repair.

In response to ultraviolet B damage, keratinocytes undergo apoptosis to eliminate damaged cells, thereby preventing tumorigenic transformation. Caffeine, the most widely consumed psychoactive substance, produces complex pharmacological actions; it has been shown to be chemopreventive in non-melamona skin cancer in mice through increasing apoptosis. Here we have investigated the molecular and cellular mechanisms in the pro-apoptotic effect of caffeine on UVB-irradiated human HaCaT keratinocytes. Pretreatment with caffeine increased UVB-induced apoptosis in HaCaT cells. Caffeine blocked UVB-induced Chk1 phosphorylation. In addition, similar to the effect of the PI3K inhibitor LY294002, caffeine also inhibited phosphorylation of AKT and up-regulation of COX-2, two critical oncogenic pathways in skin tumorigenesis. However, phosphorylation of EGFR or ERK was unaffected. Inhibiting ATR pathways by siRNA targeting ATR had little effect on UVB-induced apoptosis or AKT activation, indicating that the inhibitory effect of caffeine on apoptosis and the AKT pathway does not require the ATR pathway. Inhibiting AKT by caffeine blocked UVB-induced COX-2 up-regulation. Expression of constitutively active AKT that was not inhibited by caffeine was found to protect cells from caffeine-promoted apoptosis post-UVB irradiation, indicating that AKT is an essential inhibitory target for caffeine to promote apoptosis. Caffeine specifically sensitized cells with unrepaired DNA damage to UVB-induced apoptosis. These findings indicate that in HaCaT keratinocytes, inhibiting the AKT/COX-2 pathways through an ATR-independent pathway is a critical molecular mechanism by which caffeine promotes UVB-induced apoptosis of unrepaired keratinocytes for elimination.

Regulation of global genome nucleotide excision repair by SIRT1 through xeroderma pigmentosum C.

Disruption of the nucleotide excision repair (NER) pathway by mutations can cause xeroderma pigmentosum, a syndrome predisposing affected individuals to development of skin cancer. The xeroderma pigmentosum C (XPC) protein is essential for initiating global genome NER by recognizing the DNA lesion and recruiting downstream factors. Here we show that inhibition of the deacetylase and longevity factor SIRT1 impairs global genome NER through suppressing the transcription of XPC in a SIRT1 deacetylase-dependent manner. SIRT1 enhances XPC expression by reducing AKT-dependent nuclear localization of the transcription repressor of XPC. Finally, we show that SIRT1 levels are significantly reduced in human skin tumors from Caucasian patients, a population at highest risk. These findings suggest that SIRT1 acts as a tumor suppressor through its role in DNA repair.

UVB-Induced p21 degradation promotes apoptosis of human keratinocytes.

Skin cancer is the most common cancer in the United States. Ultraviolet B (UVB) radiation in sunlight is the major environmental factor causing skin cancer. p21, a p53-inducible protein, plays an important role in cell cycle, DNA repair, and apoptosis. Here we have investigated the effect of UVB radiation on p21 and its molecular mechanisms and function in human HaCaT keratinocytes, which we used as a premalignant cellular model because normal skin harbors numerous clones of p53-mutated keratinocytes. We found that in human HaCaT keratinocytes UVB induces rapid p21 down-regulation via a proteasomal degradation mechanism. In p53-defective HaCaT cells, the p21 protein levels remain decreased at a later time post-UVB, but in normal human and mouse epidermal keratinocytes with wild-type p53 the p21 levels are initially reduced but later increase post-UVB. These findings indicate that loss of p53 function leads to sustained p21 down-regulation in response to UVB damage. Degradation of p21 following UVB radiation does not require ATR, ATM, or both, because either the ATR/ATM inhibitor caffeine or siRNA knockdown of ATR, ATM, or both failed to reverse p21 degradation. However, inhibiting MDM2 or GSK3ß partially reduced UVB-induced p21 degradation, while inhibiting both enzymes completely prevented it. Restoring the p21 protein levels in UVB-irradiated keratinocytes reduced apoptosis. Although at the molecular level increasing p21 expression has no effect on the protein levels of the Bcl-2 family members, it enhances the activation of AKT, a critical survival pathway to protect cells from apoptosis. Our results suggest a distinct mechanism of p21 degradation in keratinocytes by UVB, and this p21 degradation may significantly enhance UVB-induced apoptosis of premalignant keratinocytes with a p53 defect to eliminate damaged cells and therefore prevent skin cancer development.

Immunosuppressive cyclosporin A activates AKT in keratinocytes through PTEN suppression: implications in skin carcinogenesis.

Non-melanoma skin cancer, the most common neoplasia after solid organ transplantation, causes serious morbidity and mortality and is related to sun exposure. Cyclosporin A (CsA) has been used widely to prevent rejection in organ transplantation. The mechanism of CsA action in causing cancer was thought to be well understood via immunosuppression. Here, we show that CsA promotes primary skin tumor growth in immune-deficient mice and keratinocyte growth in vitro. In addition, CsA enhances keratinocyte survival from removal of extracellular matrix or UVB radiation. At the molecular level, CsA increases AKT activation after serum treatment and UVB irradiation. Furthermore we found that expression of PTEN, the negative regulator of AKT activation, is significantly reduced post-CsA in human HaCaT and A431 cells and in mouse skin in vivo. CsA-induced PTEN down-regulation occurs at the transcription level and is epidermal growth factor receptor-dependent. Such PTEN suppression is required for increased AKT activation. Inhibition of AKT activation abolishes CsA-promoted growth and survival, indicating that AKT hyperactivation is essential for both growth and survival of CsA-treated cells. In addition, mTOR signaling as a known AKT downstream pathway is required for CsA-enhanced growth and survival. Taken together, we have identified the PTEN/AKT pathway as new molecular targets of CsA in epidermal keratinocytes, suggesting a previously unknown mechanism in CsA-enhanced skin carcinogenesis. Our findings challenge assumptions about how CsA-associated tumors arise in skin.

Requirement for metalloproteinase-dependent ERK and AKT activation in UVB-induced G1-S cell cycle progression of human keratinocytes.

UVB (280-315 nm) in natural sunlight represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here we demonstrate that low doses of UVB induce keratinocyte proliferation and cell cycle progression of human HaCaT keratinocytes. Different from UVA, UVB irradiation induced extracellular signal-regulated kinase (ERK) and AKT activation and their activation are both required for UVB-induced cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVB exposure and is upstream of ERK/AKT/cyclin D1 pathway activation and cell cycle progression following UVB radiation. Furthermore, metalloproteinase (MP) inhibitor GM6001 blocked UVB-induced ERK and AKT activation, cell cycle progression, and decreased the EGFR phosphorylation, demonstrating that MPs mediate the EGFR/ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB radiation. In addition, ERK or AKT activation is essential for EGFR activation because ERK or AKT inhibitor blocks EGFR activation following UVB radiation, indicating that EGFR/AKT/ERK pathways form a regulatory loop and converge into cell cycle progression following UVB radiation. Identification of these signaling pathways in UVB-induced cell cycle progression of quiescent keratinocytes as a process mimicking tumor promotion in vivo will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer.

[Gene expression profiles in different tissues of human nasopharyngeal carcinoma].

OBJECTIVE: To screen the genes that may play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). METHODS: Microdissection and cDNA genechip hybridization techniques were used to examine the differentially expressed genes in NPC tissue, the surrounding and adjacent tissues of NPC, and the nasopharyngeal inflammation tissue. The fluorescent signals on cDNA chip were scanned and the results of hybridization analyzed by image processing software. RESULTS: Many differentially expressed genes were identified between the three samples, including many different types of genes, such as those responsible for signal and protein transmission, oncogene and tumor suppression genes, immune-associated genes, apoptosis genes and DNA binding and transcription factor genes. CONCLUSION: The carcinogenesis of NPC involves many genes of a variety of types, suggesting its complex process.

[Effects of site-directed mutagenesis at amino acid residues of GATA-1b different from that of GATA-1a in Xenopus].

The GATA-1 of Xenopus (xGATA-1), which has two subtypes xGATA-1a and xGATA-1b, is a necessary factor for erythroid differentiation and maturation as similar as that of other GATA-1s. Although both xGATa-1a and xGATA-1b are able to stimulate erythropoiesis, only xGATA-1b is capable of inhibiting neurogenesis in Xenopus embryos. Compared between their structures, xGATA-1a and xGATA-1b are very similar in nucleotide and amino acids composition, but not identical. Therefore, it is responsible for studying the role of the diverse codons between the two genes, so the desired mutations: S(168), H(169) double deletion and point mutation of T(304)-->A, T(359)-->A, were introduced into xGATA-1b gene through site-directed mutagenesis. Then, mRNA from each mutant as well as wtxGATA-1b was co-injected with DN-BR mRNA or separately injected into Xenopus stage 2 embryos, and the role of mutants in erythropoiesis and neurogenesis was analyzed by using animal cap culture system. The results showed that the neural-inhibiting activity of xGATA-1b, but not hematopoiesis-inducing activity, was aborted because of deletion of Ser(168) and His(169) or point mutation of T(359)-->A. So it is demonstrated for the first time that Ser(168) and His(169) or Thr(359)in xGATA-1b may be one of the structural basis for explanting the different function between xGATA-1b and xGATA-1a.

[Analysis of characteristics of gene expression in pericancerous stromal cells of nasopharyngeal carcinoma by cDNA array].

BACKGROUND & OBJECTIVE: The mechanism of how stromal cell play an important role in nasopharyngeal carcinogenesis is now a hotspot. This study was designed to elucidate the possible mechanism of stromal cell in the occurrence and progression of nasopharyngeal carcinoma (NPC) through the analysis of the characteristics of gene expression in pericancerous stromal cells of NPC by cDNA array. METHODS: The atlas human select tumor arrays were used to compare the expression profiles between NPC tissue and NPC cell lines. RESULTS: Pericancerous stromal cells in NPC expressed at least 40 genes specifically. CONCLUSION: The specific expression of these genes in pericancerous stromal cells provides energy materials for growth of NPC cells; furthermore, it can accelerate the degradation of extracellular matrix, thus promoting the metastasis of NPC cells.

[Plerosis of cDNA array of normal human nasopharyngeal tissue and nasopharyngeal carcinoma].

OBJECTIVE: To compare gene expression gene profile of nasopharyngeal carcinoma (NPC) tissue with that of normal nasopharyngeal tissues by cDNA array and to discuss possible functions of DNA repair-related genes in NPC tissue. METHODS: After hybridization of atlas human cancer cDNA expression array 7742 - 1, atlas hybridization results were analyzed by Atlas Image 1.01 a software package. Using RT-PCR was used to confirm the results. RESULTS: Of 63 differentially expressed genes in quadrangle C including DNA damage response, repair & recombination-related genes, 6 DNA repair-related genes were up-regulated, 12 were down-regulated. CONCLUSION: DNA repair-related genes may be involved in patho-physiological process of nasopharyngeal carcinoma.

[Application of microdissection combined with RNA linear amplification].

BACKGROUND & OBJECTIVE: Microdissection has become indispensable for the selective analysis of stroma-free tumor cell. However, To obtain sufficient RNA from microdissected cells is difficult. The study was designed to seek a specific way to separate nasopharyngeal carcinoma (NPC) cells from stromal cells and to amplify the RNA from microdissected NPC cells. METHODS: NPC cells were obtained using microdissection from frozen NPC tissue sections, then RNA was extracted from the microdissected NPC cells and reverse transcribed in vitro. The expression levels of beta-actin and GADPH in amplified RNA were detected using RT-PCR. RESULTS: About 20,000-40,000 NPC cells were obtained, RNA was extracted from the cells, about 0.5-2.5 kb RNA fragments were obtained after RNA linear amplification and beta-actin and GADPH levels were integral. CONCLUSION: Microdissection combined with RNA linear amplification can be used to successfully obtain pure NPC cells, the integrity of amplified RNA is good and can be used in further research.