NKX6.3 protects against gastric mucosal atrophy by downregulating β-amyloid production

Figure 2 NKX6. 3 inhibits amyloid β accumulation by regulating apolipoprotein E, β-, and γ-secretase. A: Western blot analysis showing increased expression of apolipoprotein E (ApoE), amyloid precursor protein (APP), amyloid β (Aβ), β-secretase 1 (Bace1), low-density lipoprotein receptor (LDLR), nicastrin (NCT), high mobility group box1 (HMGB1), and receptor for advanced glycosylation end product (RAGE) proteins but decreased expression of presenilin 1 (PSN1)- C-terminal (CTF) protein in NKX6.3 depleted HFE-145 cells. Interestingly, HFE-145^shCtrl cells expressed only Aβ monomer whereas NKX6.3 depleted HFE-145^shNKX6.3 cells induced production of oligomers of Aβ and increased expression of Aβ monomer. B: In immunoprecipitation assay, γ-secretase complex including PSN1-CTF and 2-CTF, NCT, and presenilin enhancer 2 (PEN2) was stably formed, binding to Bace1 protein only in NKX6.3 depleted HFE-145^shNKX6.3 cells. C: Immunofluorescence images showing strong expression of Aβ protein in the cytoplasm of NKX6.3 depleted HFE-145^shNKX6.3 cells. D: In Aβ42 enzyme-linked immunosorbent assay, NKX6.3 depletion significantly increased concentrations of aggregated Aβ in both cell lysate and culture media. Shown are the means ± SEM from three experiments. Superscript letter significant difference (^aP < 0.0001). E: Increased mRNA expression of ApoE and Bace1 genes was detected in NKX6.3 depleted HFE-145^shNKX6.3 cells. Shown are the means ± SEM from three experiments. Superscript letter represents significant difference (^bP < 0.005 and ^cP < 0.0001). F: In chromatin immunoprecipitation assay, the binding capacity of NKX6.3 to promoter regions of ApoE and Bace1 genes was dramatically decreased. Shown are the means ± SEM from three experiments. Superscript letter represents significant difference (^dP < 0.005 and ^eP < 0.0001). G: When we treated HFE-145^shCtrl and HFE-145^shNKX6.3 cells with Aβ 1-42 recombinant protein (rAβ 1-42), expression levels of ApoE and oligomeric forms of Aβ were makedly increased in NKX6.3 depleted HFE-145^shNKX6.3 cells. CHAP: Chaps buffer; TX: Triton-100; ApoE: Apolipoprotein E; APP: Amyloid precursor protein; Aβ: Amyloid β; Bace1: β-secretase 1; LDLR: Low-density lipoprotein receptor; NCT: Nicastrin; HMGB1: High mobility group box1; RAGE: Receptor for advanced glycosylation end product; PSN1: Presenilin 1; CTF: C-terminal; PEN2: Presenilin enhancer 2; rAβ 1-42: Aβ 1-42 recombinant protein.