rhHAPLN1 induces selective increases in TGF-β RI and p-Smad3 levels with concurrent increases in SIRT1 and p300 and decreases in MMP-9 and Ac-p300 levels. Human alveolar epithelial cells (HAECs) (Passage 15) cells were seeded at 1 × 10⁵ cells/6-well plate culture with CHECM (Cell Biologics). Protein levels were analyzed by western blotting as described in Materials and Methods section. (A) rhHAPLN1 (25 and 50 ng/ml) was treated for 24 h. (B) HAECs were treated with rhHAPLN1 (20 and 100 ng/ml) and/or HA (>2,500 kDa) (20 and 100 µg/ml) at the indicated concentrations for 24 h in serum-free condition. (C) At 24 h after rhHAPLN1 treatment, TGF-β1 (10 ng/ml) was treated for 0, 15, 30, and 60 min. The protein levels of TGF-β RI, TGF-β RII, Smad2/3, p-Smad2, p-Smad3, Ac-p300, p300, SIRT1, MMP9, and GAPDH were determined by western blotting. Protein samples (15 µg per lane) were loaded for immunoblot as described in Materials and Methods section. The results (mean ± SEM) represent three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus Control or non-rhHAPLN1 group. ns, no significance. rhHAPLN1, recombinant human hyaluronan and proteoglycan link protein 1; TGF-β RI, transforming growth factor β receptor 1; p-Smad3, phosphorylated Smad3; SIRT1, sirtuin 1; MMP9, metalloproteinase-9; Ac-p300, acetylated p300.|@|~(^,^)~|@|Rescue effects of rhHAPLN1 on protein levels of TGF-β RI, SIRT1/2/6, and CARM1 reduced by cell-damaging insults, and its ROS scavenging effects. Human alveolar epithelial cells (HAECs) (Passage 3) were seeded at 1 × 10⁵ cells/6-well plate culture with CHECM (Cell Biologics). (A) At 24 h after porcine pancreatic elastase (PPE) (15 mU/ml) pretreatment, rhHAPLN1 (25 and 50 ng/ml) was treated for 24 h. The levels of TGF-β RI, SIRT1, SIRT2, SIRT6, CARM1, and GAPDH were analyzed by western blotting as described in Materials and Methods section. (B) HAECs (Passage 16) were transfected with either siNC or siCARM1 with CHECM. At 5 h after CARM1 siRNA pretreatment, CHECM was replaced with a complete medium for 19 h. At 24 h after CARM1-knockdown, following pre-treatment of 300 µM H₂O₂ for 1 h, rhHAPLN1 (25 ng/ml) was treated for 24 h. (C) HAECs (Passage 3) were exposed to 300 µM H₂O₂, 15 mU/ml PPE, 20 ng/ml IL-1β, and 1 µg/ml LMW-HA for 1 h and incubated for 24 h in the presence or absence of rhHAPLN1. ROS was measured as the fluorescence intensity of DCFDA using FACS. (D) HAECs were treated with rhHAPLN1 (25 and 50 ng/ml) at the indicated concentrations for 24 h in serum-free conditions. (E) At 1 h after PPE (15 mU/ml) pretreatment, rhHAPLN1 (25 and 50 ng/ml) was treated for 24 h. The protein levels of TGF-β RI, SIRT1, SIRT2, SIRT6, CARM1, Nrf2, p53, p21, p16, and GAPDH were determined by western blotting. Protein samples (15 µg of protein per lane) were loaded for immunoblot as described in Materials and Methods section. The results (mean ± SEM) represent three independent experiments (n = 3). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus Control. *P < 0.05, **P < 0.01, ***P < 0.001 versus positive Control (insults treatments). ns, no significance. rhHAPLN1, recombinant human hyaluronan and proteoglycan link protein 1; TGF-β RI, transforming growth factor β receptor 1; SIRT1, sirtuin 1; CARM1, co-activator-associated arginine methyltransferase-1; ROS, reactive oxygen species.|@|~(^,^)~|@|The effects of CD44 deficiency on the levels of TGF-β RI, SIRT1/2/6, and p-NF-κB in various cell-damaging insults-exposed HAECs, and rescue effects exerted by rhHAPLN1. HAECs (Passage 13) were seeded at 1 × 10⁵ cells/6-well plate culture with CHEM (Cell Biologics). At 4 h after CD44 siRNA pretreatment, CHECM was replaced with a complete medium for 20 h. (A) rhHAPLN1 (25 and 50 ng/ml) were treated for 24 h. In addition, to test the reversal effects of rhHAPLN1 on such changes in levels induced by cell-damaging insults, at 24 h after the CD44-knockdown, H₂O₂ 300 µM (B), IL-1β 20 ng/ml (C), and low molecular weight hyaluronan (LMW-HA) 1 µg/ml (D) were pre-treated for 1 h, followed by rhHAPLN1 (25 ng/ml) treatment for 24 h. The protein levels of CD44, TGF-β RI, SIRT1, SIRT6, NF-κB, p-NF-κB, and GAPDH were determined by western blotting. Protein samples (15 µg per lane) were loaded for immunoblot as described in Materials and Methods section. The results (mean ± SEM) represent three independent experiments (n = 3). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus Control. *P < 0.05, **P < 0.01, ***P < 0.001 versus positive Control (insults treatments). ns, no significance. TGF-β RI, transforming growth factor β receptor 1; SIRT1, sirtuin 1; p-NF-κB, phosphorylated NF-κB; HAECs, human alveolar epithelial cells; rhHAPLN1, recombinant human hyaluronan and proteoglycan link protein 1.|@|~(^,^)~|@|rhHAPLN1 reduced significantly the length of MLI of alveoli as an indicative of the mouse senile emphysema. Five young male C57BL/6J mice aged 6 to 10 weeks and two groups of five old male mice aged 19 to 20 months were used. Young mice and one group of old mice were treated with phosphate-buffered saline (PBS) as Control groups. One group of the old mice was intraperitoneally injected with rhHAPLN1 (0.1 mg/kg) in PBS once every three days for nine times and then euthanized after 1 day of the final injection. The lungs were harvested, parasagittal sectioned, immersed in formalin, and paraffin blocks were made. The lung tissues were cut into 5 µm and stained with H&E. (A) Pictures of representative histological sections of the lungs were taken with a 200× optical microscope. (B) MLI (mean ± SEM) of alveolar septae were measured in the lungs of five young mice, the control group of five old mice, and rhHAPLN1-treated group of five old mice. The Stitcher ImageJ plugin program was used (Preibisch et al., 2009). *P < 0.05, ***P < 0.001. Scale bars = 100 µm. rhHAPLN1, recombinant human hyaluronan and proteoglycan link protein 1; MLI, mean linear intercepts.|@|~(^,^)~|@|rhHAPLN1 reduced significantly the length of MLI of alveoli as an indicator of the mouse PPE-induced COPD model. Male C57BL6/N mice aged 6 to 10 weeks were used as the experimental model. Mice were divided into four groups with six mice in each group in each of two independent experiments. In the 1st experiment, the groups were Normal, Control, rhHAPLN1 (0.00033% w/v), and high molecular weight hyaluronan (HMW-HA) groups (0.0057% w/v) (A and B), and in the 2nd experiment, groups were Normal, Control, rhHAPLN1-(a) (0.0001% w/v), and rhHAPLN1-(b) (0.0005% w/v) (C and D). In each experiment, to induce COPD in the three groups except Normal, on the day before the exposure to the test samples, PPE (30 µl/6 Unit/time) was injected into the oral airway with a 0.9 mm × 50 mm-oral zonde as a method of alveolar inhalation, and rhHAPLN1 or HMW-HA solubilized in vehicle saline was administered on the second day for 1 h daily and five times per week for 21 days, and then mice were euthanized after 1 day of the final exposure. The lungs were harvested, parasagittal sectioned, immersed in formalin, and paraffin blocks were made. The lung tissues were cut into 5 µm and stained with H&E. Pictures of representative histological sections of lungs were taken with a 200× optical microscope (A and C). MLI (mean ± SEM) of alveolar septae were measured in the lungs of six Normal mice, Control group of six saline-treated mice, and the rhHAPLN1-treated group of six mice. The Stitcher ImageJ plugin program was used (Preibisch et al., 2009) (B and D). Compared with those of each Control group, the results from 1st experiment showed that rhHAPLN1 significantly reduced the enlargement of alveoli as an indicative of emphysema: 0.00033% rhHAPLN1 group by 95% [(21.2 – 13.8)/21.2 – 14.5) × 100]; 0.0057% HMW-HA by 73.0% [(21.2 – 13.8)/21.2 – 15.8) × 100] (B), while 2nd experiment showed 0.0001% rhHAPLN1-(a) by no significance; 0.0005% rhHAPLN1-(b) by 87% [(45.1 – 26.2)/(45.1 – 23.4) × 100] (D). *P < 0.05, **P < 0.01, ***P < 0.001. ns, no significance. Scale bars = 100 µm. (E) A proposed model showing a cross-talk between HAPLN1, CD44, TGF-β signaling, CARM1, sirtuins (SIRT1/2/6), and p300 for explaining the rhHAPLN1-mediated mitigation of COPD. Arrow lines indicate pathways which have been evidenced by our present data and previous studies; arrow dashed lines indicate pathways which remain elucidated through further studies. rhHAPLN1, recombinant human hyaluronan and proteoglycan link protein 1; MLI, mean linear intercepts; PPE, porcine pancreatic elastase; COPD, chronic obstructive pulmonary disease; HA, hyaluronan; ROS, reactive oxygen species; TGF-β RI, transforming growth factor β receptor 1; p-Smad3, phosphorylated Smad3; Nrf2, nuclear factor erythroid 2-related factor 2; CARM1, co-activator-associated arginine methyltransferase-1; Ac-p300, acetylated p300; p-NF-κB, phosphorylated NF-κB; MMP9, metalloproteinase-9.

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