Stiff Matrix Instigates Type I Collagen Biogenesis by Mammalian Cleavage Factor I Complex-Mediated Alternative Polyadenylation

Alternative polyadenylation (APA) is a widespread and important mechanism in regulation of gene expression. Dysregulation of the 3' UTR cleavage and polyadenylation represents a common characteristic among many disease states including lung fibrosis. In this study, we investigated the role of mammalian cleavage factor I (CFIm)-mediated APA in regulating the extracellular matrix production in response to mechanical stimuli from stiffened matrix simulating the fibrotic lungs. We found that stiff matrix downregulates expression of CFIm68, CFIm59 and CFIm25 subunits, and promotes APA in favor of the proximal poly(A) site usage in the 3' UTRs of type I collagen (COL1A1) and fibronectin (FN1) in primary human lung fibroblasts. Knockdown and overexpression of each individual CFIm subunit demonstrated that CFIm68 and CFIm25 are indispensable attributes of stiff matrix-induced APA and overproduction of COL1A1, whereas CFIm does not appear to mediate stiffness-regulated FN1 APA. Furthermore, expression of the CFIm subunits is associated with matrix stiffness in vivo in a bleomycin-induced mouse model of pulmonary fibrosis. These data suggest that stiff matrix instigates type I collagen biogenesis by selectively targeting mRNA transcripts for 3' UTR shortening. The current study uncovered a potential mechanism for regulation of the CFIm complex by mechanical cues under fibrotic conditions.


Isolation of primary lung fibroblasts
Lungs were minced in sterile phosphate-buffered saline and tissue pieces were placed in 100mm tissue culture dishes containing Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 1% penicillin/streptomycin/glutamine, nonessential amino acids, and sodium pyruvate (supplemented DMEM). Medium was replenished every 3 d. After 14 d, cells growing out of the explants were trypsinized and plated in supplemented DMEM. Lung fibroblasts were used between passages 6 and 10.

Quantitative real-time PCR
Total RNA was isolated using TRIzol reagent (ThermoFisher Scientific, Waltham, MA). 1 μg total RNA was reversely transcribed into cDNA with a cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA). Quantitative PCR reactions were carried out in a Bio-Rad iCycler (Bio-Rad Laboratories, Hercules, CA). Each sample was run in triplicate. Relative quantification was calculated using the comparative CT method 1 . Delta CT values of target gene were normalized to GAPDH and subjected to statistical analysis. The ratio of distal transcripts/common transcripts was calculated as ΔCT = (CTdistal -CTGAPDH) -(CTcommon -CTGAPDH) = CTdistal -CTcommon.

Construction of CFIm subunit-expressing lentiviruses and cell infection
The full-length cDNAs of human CFIm68, CFIm59, and CFIm25 corresponding to NCBI Virus-containing conditioned medium was harvested 72 hrs after transfection, filtered, and used to infect recipient lung fibroblasts in the presence of 8 μg/mL polybrene (Sigma, Louis, MO).

siRNA-mediated gene knockdown
100 pmol CFIm siRNAs or scrambled siRNAs were transfected into 1 x 10 6 lung fibroblasts by a Nucleofector device (Lonza, Basel, Switzerland) according to the manufacturer's protocol.
Transfected cells were cultured in DMEM supplemented with 10% FBS for 72 h before harvesting.

Immunoblot and densitometry analysis
Cell lysates containing 10 -40 µg total proteins were loaded onto SDS-polyacrylamide gels under reducing conditions. After electrophoresis, proteins were electrophoretically transferred from the gels to nitrocellulose at 100 V for 1.5 hr at 4°C. Membranes were blocked in casein solution (1% casein, 25 mM Na2HPO4, pH 7.1) for 1 hr at room temperature. Primary antibodies were diluted in TBS-T and casein solution (1:1) at a working concentration recommended by manufactures. Membranes were incubated with primary antibodies at room temperature for 1 hr.
After extensive washing, membranes were incubated with peroxidase-conjugated secondary antibodies (0.1 µg/ml) diluted in TBS-T for 1 hr at room temperature. Immunodetection was carried out by chemiluminescence. Blot images were scanned. Bands were quantified by ImageJ (NIH, Bethesda).

AFM measurements of lung tissue elasticity
AFM probes were composed of silicon nitride coating with Au (TR400PB, spring constant ranging 10-50pN/nm) (Oxford Instruments Asylum Research Inc, Goleta, CA). The AFM system was calibrated according to the manufacturer's instruction before each indentation measurement, and the cantilever spring constant was confirmed by the thermal fluctuation method. Force-indentation profiles were acquired at an indentation rate of 20 μm/s separated by 4 μm spatially in a 16 × 16 sample grid covering a 20 × 20-μm area. The elasticity (Young's modulus) at each point on the grid was calculated from fitting force-indentation data using a Hertz-based model: F=2Eδ 2 tan(α)/π(1-ν 2 ), where indentation force (F) was calculated by using Hooke's law (F = κΔx), where κ and Δx denote the AFM probe's spring constant and the probe's measured deflection, respectively. The indentation depth (δ) is calculated from the difference in the z-movement of the AFM piezo and the deflection of the probe. E is the elastic modulus of lung tissues being studied, and ν denotes the Poisson ratio (0.4). α represents the shape of the probes that were considered to be conical with an approximated half-angle of 35 degrees in this report. A contour map was plotted to visualize spatial patterns of elasticity collected in Force-Map mode using the Matlab R2019a (MathWorks, Inc., Natick, MA).

Lung histology and immunofluorescent staining
Picrosirius red staining was performed to identify collagen content in mouse lung tissue samples according to the manufacturer's recommendation (Abcam, Cambridge, UK). Digital images of the stained sections were captured using an Olympus light microscopy.. Images were obtained with a Nikon Eclipse TE 300 microscope equipped with Spot Insight CCD camera and MetaMorph software version 6.2 r4 (Universal Imaging Corp., Downington, PA).