Alterations in DICER and miRNA levels with cigarette smoke

1

A microRNA processing defect in smokers' macrophages is linked to SUMOylation of the

endonuclease DICER

Thomas J. Gross, Linda S. Powers, Ryan L. Boudreau, Brandi Brink, Anna Reisetter,

Khushboo Goel, Alicia K. Gerke, Ihab H. Hassan and Martha M. Monick

From the Department of Medicine, Carver College of Medicine,

University of Iowa, Iowa City, Iowa, 52242

* Running title: Alterations in DICER and miRNA levels with cigarette smoke

To whom correspondence should be addressed: Thomas J. Gross, Division of Pulmonary, Critical

Care, and Occupational Medicine, University of Iowa, Iowa City, IA 52242, Phone: (319) 356-6268,

Email: thomas-gross@uiowa.edu

Keywords: COPD, DICER, inflammation, miRNA, macrophages

Background: Smoking causes a global down

regulation in alveolar macrophage miRNA

expression.

Results: Cigarette smoke exposure modifies the

RNA endonuclease DICER, resulting in a

microRNA-processing defect.

Conclusion: Cigarette smoke alters alveolar

macrophage microRNA expression, in part, by

SUMOylation of DICER.

Significance: This is the first description of an

environmental exposure causing changes in

microRNA expression via post-translational

modification of Dicer.

ABSTRACT

Despite the fact that alveolar macrophages

play an important role in smoking-related disease,

little is known about what regulates their

pathophysiologic phenotype. Evaluating smoker

macrophages, we found significant down

regulation of multiple microRNAs (miRNAs).

This project investigates the hypothesis that

cigarette smoke alters mature miRNA expression

in lung macrophages by inhibiting processing of

primary miRNA transcripts. Studies on smoker

alveolar macrophages showed a defect in miRNA

maturation. Studies on the miRNA biogenesis

machinery led us to focus on the cytosolic RNA

endonuclease, DICER. DICER cleaves the stem-

loop structure from pre-miRNAs, allowing them to

dissociate into their mature 20-22 nucleotide

single stranded form. DICER activity assays

confirmed impaired DICER activity following

cigarette smoke exposure. Further protein studies

demonstrated a decreased expression of the native

217 kD form of DICER and an accumulation of

high molecular weight forms with cigarette smoke

exposure. This molecular mass shift was shown to

contain SUMO moieties and could be blocked by

silencing RNA directed at the primary

SUMOylating ligase, Ubc9. In determining the

cigarette smoke components responsible for

changes in DICER, we found that N-acetyl-

cysteine (NAC), an anti-oxidant and anti-

aldehyde, protected DICER protein and activity

from cigarette smoke extract (CSE). This massive

down regulation of miRNAs (driven in part by

alterations in DICER) may be an important

regulator of the disease promoting macrophage

phenotype found in smoker's lungs.

INTRODUCTION

COPD is characterized by inflammation in

both small airways and alveoli eventually leading

to irreversible airflow limitation and parenchymal

emphysema. Lung pathology in patients with

COPD has been largely attributed to neutrophil

elastases and macrophage proteinases (1,2). While

multiple lung cell types contribute to the

pathogenesis of COPD, evidence strongly supports

a key role for alveolar macrophages (3-5). Inhaled

tobacco smoke triggers disease-relevant changes

in alveolar macrophage gene expression (i.e.

MMP12) (6,7). While the regulation of disease-

relevant genes is complex and multifactorial,

increasing experimental evidence suggests that

macrophage gene expression is strongly

influenced by miRNAs (8,9). Our laboratory has

http://www.jbc.org/cgi/doi/10.1074/jbc.M114.565473The latest version is at JBC Papers in Press. Published on March 25, 2014 as Manuscript M114.565473

Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Alterations in DICER and miRNA levels with cigarette smoke

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demonstrated that cigarette smoking is associated

with an impressive decrease in alveolar

macrophage miRNA expression (10). Detailing

the mechanism(s) behind this significant decrease

in miRNA levels could shed insight into the

pathophysiology of smoking-related lung diseases

and improve understanding of the adverse health

effects of cigarettes in general.

The role of miRNA levels in regulating

cellular genomic tone is an area of active

investigation (11-13). Primary miRNAs are

transcribed in the nucleus and processed into

precursor miRNA (pre-miRNA) by a nuclear

endoribonuclease, Drosha, in complex with a

double-stranded RNA binding protein, Pasha

(known as DGCR8 in vertebrates). The pre-

miRNA is an approximately 70 base pair double-

stranded RNA stem-loop structure that contains

the imbedded 22-nucleotide sequence

complementary to the mRNA target of interest.

Pre-miRNAs are exported from the nucleus where

the cytoplasmic endoribonuclease, DICER, excises

the stem-loop yielding a 20 to 25 base-pair RNA

duplex (14,15). In the cytosol, DICER, in complex

with other protein components (transactivating

response RNA-binding protein TRBP, protein

kinase RNA activator PACT, and Argonaut

protein Ago2) binds the duplex, slices out the anti-

complementary “passenger” RNA strand, and

targets it for destruction. The mature miRNA and

processing proteins form an RNA-induced

silencing complex (RISC), which represses

translation or enhances degradation of the targeted

complementary mRNA. Thus, up or down

regulation of an miRNA can result in specific

regulation of gene expression. The miRNA

biogenesis pathway has several potential points for

regulatory control. In this manuscript, we present

evidence that cigarette smoke exposure leads to a

post-translational modification of DICER,

resulting in decreased ribonuclease activity and

contributing to a generalized decrease in miRNA

expression in smokers' alveolar macrophages.

EXPERIMENTAL PROCEDURES

Reagents and antibodies: Antibodies used in

this study were obtained from a variety of sources:

anti-HA, Roche Biochemical Laboratories,

#11867423001; anti-DICER, Abcam, #ab14601;

anti-PACT, Abcam, #ab75749; anti-TRBP,

Abcam, #ab42018; anti actin, Abcam, #ab8226;

anti-UBC9, Abcam, ab21193; anti-SUMO 2/3,

Abcam, #ab3742; anti-ubiquitin, Cell Signaling,

#3936. Horseradish peroxidase conjugated anti-

rabbit (sc-2004), anti-mouse (sc-2005) and anti-

goat (sc-2020) antibodies were all obtained from

Santa Cruz Biotechnology. Culture media used in

experiments included serum free RPMI tissue

culture media with Glutamax (Invitrogen, 61870-

036) and DMEM (Invitrogen, 11095) with 10%

FCS (Hyclone Fetal Bovine Serum, SH30071).

Ethics statement: The University of Iowa

Institutional Review Board approved all

procedures and protocols described in this

communication. Written informed consent was

obtained and all clinical investigation has been

conducted according to the principles expressed in

the Declaration of Helsinki.

Alveolar macrophage donors: Subjects were

recruited from the community through the Iowa

Institute for Clinical and Translational Science

(ICTS) Clinical Core. Inclusion criteria for

smoking subjects required at least a 10 pack-year

history of ongoing cigarette smoking, while the

nonsmoker control subjects were self-reported

never smokers. Subjects were excluded if they had

any significant co-morbid conditions such as

pregnancy or other acute or chronic disease such

as pre-existing asthma, interstitial lung disease or

cardiovascular disease. Subjects were also

excluded if a baseline spirometry revealed the

forced expiratory volume in the first second was

less than 60% of the predicted value based on

National Health and Nutrition Examination Survey

III data set. None of the subjects were using

inhaled corticosteroids.

Bronchoalveolar lavage: After informed

consent was obtained, subjects underwent standard

flexible bronchoscopy (16). Local anesthesia with

lidocaine instillation into the upper airway was

followed by bronchoalveolar lavage (BAL)

whereby 20 ml of normal saline was instilled into

a tertiary bronchus up to five times in three

different lung segments. The first collection out of

five was discarded to avoid possible contamination

with upper airway secretions or lidocaine. The

remaining lavage was filtered through sterile

gauze and centrifuged at 200xg for 5 minutes to

pellet cellular material. The resulting pellet was

suspended in phosphate buffered saline (PBS) and

centrifuged at 200xg for 5 minutes. A sample of

the cells were labeled with Wright stain and

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Alterations in DICER and miRNA levels with cigarette smoke

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microscopically examined to determine the

proportion of the cells that were macrophages (17-

20). Aliquots of 5x106

cells were stored at -80°C

until RNA isolation procedure was performed.

Cell yields from bronchoscopy of nonsmokers

averaged 25±3 x 106 cells and for smokers

averaged, 67±4 x 106. In all three cohorts, the

procedure generated a relatively pure population

of alveolar macrophages with fewer than 5%

neutrophils or lymphocytes in the bronchoalveolar

lavage fluid.

CSE production and exposure: Cigarette

smoke extract was prepared as previously

described (21,22). CSE solutions were prepared

using a modification of the method of Blue and

Janoff (23). 10 mls of sterile serum free RPMI

tissue culture media with Glutamax was drawn

into a 50-ml plastic syringe. Then, 40 ml of

cigarette smoke was then drawn into the syringe

and mixed with the medium by vigorously shaking

them together. The smoke was expelled and the

process repeated until one cigarette was used up.

The generated CSE solution was filtered (0.22 μm)

to remove large particles (24,25). The resulting

solution was designated a 100% CSE solution and

was used immediately after generation. For the

CSE dialysis experiment, we dialyzed 5% CSE

overnight using Thermo Scientific’s 2000 and

10,000 MWCO Slide-A-Lyzer Dialysis Cassette

GP cassettes. RNA isolation: RNA was isolated from

alveolar macrophages using the mirVana miRNA

Isolation kit (Applied Biosystems (ABI)). The

quantity and quality of the RNA samples was

assessed using an Experion Automated

Electrophoresis Station (Bio-Rad). The RNA

quality indicator was above 8 for all samples

where values of greater than 8 indicate primarily

intact RNA on a scale of 1-10. After preparation,

RNA samples were stored at -80° C until use.

miRNA expression analysis: RNA from

alveolar macrophages of nonsmokers and active

smokers was reverse transcribed with MultiScribe

Reverse Transcriptase (ABI) using Megaplex

Primers version 2.0 (ABI). Changes in miRNA

expression were then determined using human

TaqMan Low Density Arrays version 2.0 (ABI).

Ct values calculated using SDS version 2.4 (ABI)

were exported to the Partek Genomics Suite to

calculate smoker-to-nonsmoker expression ratios.

Robust multiarray averaging (RMA)-normalized

data were subjected to an ANOVA model with

linear contrasts to calculate p-values. Individual

miRNA-specific assays were done using ABI kits

specific for the mature miRNA or for the primary

transcript (before processing).

mRNA expression analysis: Genome-wide

macrophage mRNA expression was obtained

using the GeneChip Human Exon 1.0 ST Arrays

(Affymetrix). Generation of labeled cDNA,

hybridizations, and scanning of the microarray

were performed under contract by the University

of Iowa DNA facility. The resulting data were

analyzed using the Partek Genomics Suite version

6.5 (Partek). The data were assessed for quality

and subjected to RMA normalization. The

normalized data were then analyzed using an

ANOVA model with linear contrasts to calculate

p-values and smoker-to-nonsmoker expression

ratios. The false discovery rate (FDR) step-up

method (26) was applied to correct for multiple

testing. The expression data has been deposited in

NCBI Geo repository (GSE34517). To generate

the data shown in figure3E, we mined RNA array

data performed on alveolar macrophages from 43

nonsmokers and 42 smokers. The log 2 values

obtained by the initial Partek analysis for the gene

DICER are shown in an individual value plot.

Possible significance between the two groups was

assessed by Student’s t test.

Whole cell protein isolation: Whole cell

protein was obtained by lysing the cells on ice for

20 minutes in 200 l of lysis buffer (0.05 M Tris

pH 7.4, 0.15 M NaCl, 1% NP-40, with added

protease and phosphatase inhibitors: 1 protease

Minitab (Roche Biochemical)/10 ml and 100 ul

100X phosphatase inhibitor cocktail (Calbiochem)

/10 ml. The lysates were sonicated for 20 seconds,

kept at 4o for 30 minutes, spun at 15,000 g for 10

minutes and the supernatant saved. Protein

determinations were made using the Bradford

Protein assay from Bio-Rad. Cell lysates were

stored at –70o until use.

Western analysis: Whole cell protein was

obtained as previously described (27). Following

protein purification, Western analysis was

performed following protocols as described by

Reisetter et al (16). The following antibodies were

used: anti-HA, Roche Biochemical Laboratories,

#11867423001; anti-DICER, Abcam, #ab14601;

anti-PACT, Abcam, #ab75749; anti-TRBP,

Abcam, #ab42018; anti actin, Abcam, #ab8226;

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Alterations in DICER and miRNA levels with cigarette smoke

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anti-UBC9, Abcam, ab21193; anti SUMO 2/3,

Abcam, #ab3742; and anti-ubiquitin, Cell

Signaling, #3936.

Immunoprecipitation (IP): IP of DICER was

performed on alveolar macrophages or THP-1

cells. Immunoprecipitation of cellular protein was

performed using the protein G Dynabead

immunoprecipitation kit (Invitrogen) as per the

manufacturer's instructions. Immunoprecipitation

was used in the protein modification assays and in

the DICER activity assay. DICER was pulled

down using an antibody to DICER (Abcam,

#ab14601).

In vitro DICER activity assay: An in vitro

DICER activity assay was used to test the effect of

smoking on DICER activity (28). Briefly, whole

cell protein extracts were adjusted to 1 mg/ml and

25 ul of the lysate incubated with 125 pmoles of a

27mer RNA duplex DICER substrate (DsiRNAs,

IDT) and incubated at 37oC for 8 hours. To

evaluate the conversion of 27mer dsRNA substrate

to 21mer dsRNA product, 2.5 ul of extract post

incubation was resolved on a 3.5-20% non-

denaturing PAGE gel and visualized with SYBR

green fluorescent dye. DICER activity was

calculated based on the percentage of 27mer

converted to a 21mer RNA strand. The fluorescent

image from a BioRad VersaDoc Imaging System

was converted to a tiff file and analyzed for pixels

per band using Image J, a public domain Java

image-processing program inspired by NIH Image.

In vivo DICER activity assay: To measure

Dicer activity within cells, vectors were made

expressing pre-miRNAs for two of the highly

smoking-altered miRNAs (miR-146b and miR-

708). Hela cells were transfected with the pre-

miRNA constructs and expression allowed to

proceed overnight. Cells were then exposed to

CSE for a short time (1 and 3 hours, chosen to

show Dicer altered but not depleted) and RNA

harvested. Generation of mature miRs was

determined by both qRT-PCR and Northern

analysis.

Pre-miRNA cloning: Primers for the H1

promoter that included sequence for the pre-miR

of interest were obtained from Integrated DNA

Technologies (IDT, Coralville, Iowa). These

included: H1 promoter forward primer, 5'-

ccatggaattcgaacgctgacg-3' and reverse primers for

either miR-146b 5'-aaaactcgagaaaaaacagaactgagt

ccacagggcattgctagagctcacagcctatggaattcagttctcagg

gaaagagtggtctcagacagaac-3' or miR-708, 3'-aaaact

cgagaaaaaactagaagctcacagtctagttgtgttcatgtgcaagtca

tttacccccagctagattgtaagctccttgggaaagagtggtctcagac

agaac-3'. PCR was performed using the H1

plasmid as a substrate (as described in (29)). The

resulting product (H1 promoter followed by the

specific pre-miR) was cloned into a TOPO cloning

vector (pCR 4-TOPO TA cloning vector) using the

manufacturer's protocol. Kanamycin/ampicillin

resistant clones were selected, amplified and DNA

isolated. Selected clones were grown up and

sequenced to verify the insert.

Northern analysis: Isolated RNA was run on

a 12% polyacrylamide/19:1 bis/7M urea/0.5x TBE

gel in 0.5xTBE. The gel was blotted on to

Hybond N+ membrane and probed with a P32

end-

labeled oligo designed to bind both the mature

miR and pre-miRNA sequence (miR-146b:

5'GAGCCTATGGAATTCAGTTCTCA and 3'GC

AGAACTGAGTCCACTGGGCA; miR-708:5'GA

GCCTATGGAATTCAGTTCTCA and 3’GCCAG

AACTGAGTCCACAGGGCA). For both

miR146b and miR-708, the 5' arm of the duplex

produces the major mature product. The 3' probes

were used as a control. Blots were hybridized

overnight, washed and images obtained with x-ray

film.

Ubc9 depletion with siRNA: Control siRNA

(Santa Cruz #44230) or target-specific 19-25 nt

siRNA designed to knock down Ubc9 was

transfected into Hela cells at a final concentration

of 50 nM using Lipofectamine 2000 (Invitrogen).

Transfection mixtures were assembled as

previously described (30). Cells were incubated

with 200 μl of transfection mixture and 2 ml of

antibiotic-free medium for 16-18 h. Transfection

efficiency was assessed with using a fluorescent

control siRNA (Santa Cruz #36869). Knock down

of Ubc9 was confirmed with Western Blotting. Glutathione assay: A glutathione assay was

performed on whole cell lysates using GSH-Glo

Glutathione Assay from Promega (Cat# V6911)

according to manufacturer's instructions. Statistical analysis: Unpaired Student t-test

and one-way ANOVA were used to determine

significance between experimental groups. Data

are presented as means ± SEM. The program used

for data analysis was GraphPad Prism 5.00 (San

Diego, CA).

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Alterations in DICER and miRNA levels with cigarette smoke

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RESULTS

Alveolar macrophages from chronic active

cigarette smokers demonstrate decreased mature

miRNA transcript expression when compared to

cells from nonsmokers. Our lab has previously

shown that smoker’s alveolar macrophages are

characterized by a decrease in expression of a

wide variety of miRNAs (10). In Figure 1A, we

show an analysis of the changes in miRNA that

are linked to mRNA changes between nonsmoker

and smoker alveolar macrophages These values

were extracted from the larger data set reported in

reference (10). The data for this figure was

generated from lists of altered miRNAs (> 2 fold

change) and their potential targets (determined by

Target Scan and TarBase) and significantly altered

mRNAs (q≤ 0.10) (Gene Chip Human Exon Array

1.0 ST arrays). The q value (false discovery rate)

for the mRNAs was calculated using the Partek

GS implementation of the step-up method. Gene

IDs common to both lists (miRNA targets and

mRNAs) were plotted. Each dot represents a

gene/miRNA pair as predicted by Target Scan

and/or TarBase. Of particular note are the gene/miRNA pairs

identified in the upper left and lower right

quadrants. The upper left quadrant (more mRNA

and less linked miRNA) and lower right (less

mRNA and more linked miRNA) quadrants

demonstrate multiple reciprocal relationships

between miRNA changes and mRNA changes

with smoking. Some of the genes included in the

upper left quadrant of this figure include: MMP12,

a matrix metalloproteinase linked to emphysema;

SPRY2, a possible regulator of the DICER

complex; SPARC, a regulator of extracellular

matrix; and gamma glutamylcysteine synthetase,

the first rate limiting enzyme in glutathione

synthesis. The data were significant both in the

consistency of the changes caused by smoking and

by the presence of smoking-linked changes in

genes involved in disease, i.e. MMP12, other

matrix-related genes, inflammatory genes and

angiogenesis factors (4,5,31-35). Thus, the

decrease in miRNA expression appears to have

consequences in mRNA levels and, likely, protein

expression. In order to dissect the mechanism underlying

this significant decrease in expression of miRNAs,

we selected eight miRNAs that were significantly

down regulated in our published data set (miR-

708, miR-200a, miR-210, miR-187, miR-149,

miR-429, miR-146b-3p, miR-200c) (10). Using

alveolar macrophages from four nonsmoking

control subjects and four active smokers, we

performed qRT-PCR for both mature miRNA

levels and primary transcripts. Primary miRNA

transcripts are transcribed from the genome and

include flanking regions and one or more stem

loop structures; a difference in primary miRNA

levels would imply a change in nuclear miRNA

transcription rate or alteration in primary miRNA

stability. Our assay for the mature transcript

measured only the 21/22 nucleotide miRNA; a

change in levels of only the mature miRNAs

would imply alterations in processing or stability.

Figure 1B demonstrates no decrease in the amount

of primary miRNA transcripts in smoker alveolar

macrophages. If anything, there may have been a

subtle accumulation as has been previously

reported with inhibition of DICER (36). In

contrast, consistent with our prior observations,

there was a significant comparative decrease in the

level of the mature miRNA transcripts.

Cigarette smoke exposure reduces DICER

complex activity. Because cigarette smoke-induced

alterations in miRNA levels occurred somewhere

between nuclear transcription and loading onto the

RISC-complex, we initiated studies to explore

miRNA processing activity. As these studies

required quantities of cellular protein difficult to

consistently isolate from the number of

macrophages retrieved from nonsmokers, we used

the human monocytic leukemia cell line, THP-1,

as an in vitro model. In this bioactivity assay,

FITC-labeled RNA oligomers (27mer) are

incubated with aliquots of whole cell lysates.

DICER-like activity is detected by demonstrating

cleavage of the oligomers to 21mer strands with a

band shift on gel electrophoresis. Figure 2A is a

methods development assay, showing a dose

response in whole cell lysates ability to cleave the

27mer to a 21mer. There is clear cleavage of the

27mer that varies in proportion with the total cell

protein. In Figure 2B, THP-1 cells were cultured

for 6 hours with or without 5% CSE. Whole cell

lysates were collected and an activity assay run

using 10 ug of protein. Shown in Figure 2B is a

representative gel and quantification from three

identical experiments. To more directly attribute

the measured endonuclease activity to DICER,

lysates from untreated and CSE-exposed cells

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Alterations in DICER and miRNA levels with cigarette smoke

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were subject to immunoprecipitation using a

DICER-specific antibody. As a control, parallel

samples went through the immunoprecipitation

protocol with a nonspecific IgG. 10% of the

recovered sample was incubated with a set amount

of the FITC-labeled 27mer. As shown in Figure

2C, we observed a significant decrease in DICER

activity in the IP sample recovered from smoke-

exposed cells (shown on the right is densitometry

from three separate experiments). Cigarette-smoke

exposure reduces DICER-like RNA endonuclease

activity.

To confirm the effect of cigarette smoke

exposure on mi-RNA processing in an in vivo

model, Hela cells were transfected with a construct

expressing either pre-miR-146b or pre-miR-708.

24 hours after transfection, cells were exposed to

CSE for varying times. RNA was isolated and

pre-miRNA processing assessed by both Northern

analysis and qRT-PCR. Figure 3 demonstrates

that CSE exposure leads to a decrease in

processing of pre-miRNAs into their respective

mature transcripts (Figure 3A, miR-146b and

Figure 3B, miR-708). Experiments using the 3'

Northern probes showed extremely low levels of

mature mi-RNAs, consistent with what is known

about the respective miRNAs (data not shown).

Both the Northern blot and qRT-PCR

demonstrated decreased processing of the pre-

miRNAs after CSE exposure. This in vivo assay of

pre-miRNA processing demonstrates that

cigarette-smoke exposure reduces stem-loop

cleavage activity and specific mature miRNA

levels. While we cannot exclude a concurrent

effect at the initial primary miRNA processing

step in the nucleus (e.g. DROSHA), it seems clear

that smoke exposure disrupts extra-nuclear,

terminal miRNA processing.

Cigarette-smoke decreases DICER protein

levels in exposed cells. Given the dramatic

decreases in both the levels of mature miRNA and

DICER activity after smoke exposure, we further

investigated the proteins involved in the terminal

steps of miRNA processing. Newly isolated

human alveolar macrophages retrieved from

nonsmokers were incubated for six hours in cell

culture media supplemented with freshly prepared

CSE at final concentrations ranging from 0 to 5%.

Whole cell lysates were subject to standard

Western analysis using antibodies to human

DICER, PACT, TRBP, and Ago2, with

constitutively expressed actin serving as a

control for equal protein loading. CSE-exposed

alveolar macrophages from nonsmokers

demonstrated a dose dependent change in the

molecular mass of DICER immunoreactive protein

even at very low concentrations of CSE exposure

(Figure 4A). Figure 4B demonstrates that as early

as one hour after in vitro exposure to 2% CSE,

there was a progressive loss of the 217 kD band

and appearance of multiple higher molecular

weight bands. For other proteins involved in the

cytosolic processing of pre-miRNAs, we found no

change in levels of TRBP, Ago2, or actin

demonstrating the DICER changes were not a

nonspecific cytotoxic effect from the smoke

extract. PACT was the only other complex protein

to show a change with CSE exposure. PACT also

demonstrated a relative decrease at the higher

concentrations of CSE, though this was less

pronounced than the effect on DICER (Figure 4C).

Interestingly, PACT also manifests a shift to a

higher molecular mass form after smoke exposure

similar to that seen in DICER (Figure 4C).

Because the effect of CSE on Dicer was more

pronounced that the changes in PACT, subsequent

experiments focused on DICER. Along with the

effect of CSE on DICER protein in alveolar

macrophages, similar decreases in native sized

DICER (271 kD) and the appearance of higher

molecular weight immunoreactive DICER forms

were also found in cell lysates from THP-1 and

Hela cells (Figure 4D). As the Western Blots in all

of Figure 4 were run under standard reducing

conditions, the shift to a higher molecular mass

DICER was not due to protein-protein

aggregation, but more likely a smoke exposure-

induced post-translational modification of the

DICER protein.

To assess the effect of chronic in vivo

cigarette smoke exposure on DICER protein,

whole cell lysates of alveolar macrophages were

obtained from active smokers and nonsmokers and

analyzed for DICER expression. Western Blot

analysis shown in Figures 5A & 5B confirms that

freshly isolated alveolar macrophages from active

smokers contain significantly less DICER protein

than nonsmoker’s cells. The higher molecular

weight DICER bands were also found in cells

from chronic smokers, although at seemingly

lower levels than after in vitro exposure (Figures 3

vs 4B). Expression array analysis of freshly

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Alterations in DICER and miRNA levels with cigarette smoke

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isolated alveolar macrophages showed no

difference in DICER mRNA levels between

smokers and nonsmokers confirming that smoke-

induced reductions in DICER gene transcription

were not a major contributor to our observations

(Figure 5C) Thus, cigarette smoke exposure

results in a broad decrease in mature miRNA

transcripts. This is associated with a post-

translational modification resulting in functional,

quantitative and qualitative changes in the DICER

protein.

Cigarette-smoke exposure induces post-

translational SUMOylation of DICER. The

dramatic change in apparent molecular mass on

reducing gel analysis suggested a post-

translational modification. Previously work from

out lab identified a block in the autophagy

pathway of smoke-exposed alveolar macrophages

with accumulation of high-molecular mass protein

complexes targeted for degradation. These

complexes contained both ubiquitin and SUMO

2/3 polymers. This directed us to explore whether

this high molecular mass DICER also contained

ubiquitin or SUMO 2/3 adducts.

Freshly isolated alveolar macrophages were

held in culture with or without media

supplemented with CSE. Equal amounts of protein

from whole cell lysates were subjected to

immunoprecipitation with anti-DICER antibody or

a control IgG. The proteins retrieved were

separated using reducing gel electrophoresis and

Western blots probed with anti-DICER, anti-

SUMO, or anti-ubiquitin antibodies. As seen in

Figure 6A, CSE resulted in a notable reduction in

immunoprecipitable DICER with appearance of

faint higher molecular mass bands; no similar

proteins were retrieved using a nonspecific control

antibody. When anti-DICER immunoprecipitates

were probed with antibody recognizing SUMO

2/3, there was considerable SUMO

immunoreactivity clearly detected within the high

molecular mass bands (Figure 6A). Interestingly,

although there was a reduction in total DICER

protein after CSE exposure, the SUMO 2/3

antibody suggested much of this native DICER

(217 kD) was also SUMOylated. Antibody

recognizing ubiquitin moieties also detected

immunoreactivity in the high molecular mass

DICER forms, albeit at comparatively lower levels

than seen with anti-SUMO 2/3.

DICER contains several candidate lysine

SUMOylation sites (see Supplement 1). We next

asked if blocking a key enzyme in the

SUMOylation cascade would alter the appearance

of HMW DICER forms after cigarette-smoke

exposure. The SUMO conjugating (E2) enzyme

Ubc9 catalyzes the formation of isopeptide bonds

between the C-terminus of SUMO and the amino

group of lysine in the target protein. Hela cells

were treated with varying concentration of

silencing RNA (siRNA) specific to Ubc9 or a

control siRNA and then exposed to CSE. Whole

cell lysates were then extracted and analyzed by

Western blotting. As demonstrated in Figure 6C,

the siRNA specific for Ubc9 reduced levels of

immunoreactive Ubc9 in control and CSE-exposed

cells. CSE exposure reduced levels of

immunoreactive DICER and again resulted in the

appearance of higher molecular mass forms in the

cells not exposed to smoke. Interestingly, though

the Ubc9 siRNA attenuated the CSE-induced

change in molecular mass of DICER; it did not

appear to block the reduction in immunoreactive

native DICER (217 kD). The reason for this

unexpected result is part of ongoing studies in the

lab. We also found that smoke exposure resulted

in minor increases in Ubc9 mass detected by

Western blotting (Figure 6B) that might account

for some SUMOylation change. This finding

certainly does not exclude a concurrent smoke-

induced alteration in Ubc9 activity (54, 55) and/or

effects on function of SUMOlase enzymes.

As a composite, these data show that cigarette

smoke exposure results in post-translation

modification of DICER protein that is due, at least

in part, to changes in SUMOylation.

Cigarette smoke extract induced DICER

modification is blocked by NAC and reproduced

by acrolein. Cigarette smoke contains hundreds of

known toxic chemicals that exist in both vapor and

particulate phases. As our cigarette smoke extract

was generated using an aqueous-based medium,

we knew the target compound was water-soluble.

Figure 7A demonstrates that overnight incubation

does not impact the ability of CSE to induce a shift

to higher molecular mass forms of DICER in

THP-1 cells. The DICER modifying activity is lost

when CSE is dialyzed overnight using membranes

with molecular mass cutoff > 2000 kD. Thus, the

active ingredient(s) in CSE are low molecular

weight, water soluble, and stable over many hours.

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As many of the toxic compounds generated

by the combustion of tobacco are potent oxidants,

we examined whether DICER could be protected

from CSE effects using the potent cellular

antioxidant, NAC. Cells were incubated with or

without 2 % CSE as per prior experiments and the

media concurrently supplemented with NAC at a

final concentration of 2 mM for 6 hours. Whole

cell lysates were subjected to Western blotting and

probed with anti-DICER antibody. As seen in

Figure 7B, despite the brief incubation period,

NAC supplementation increased intracellular

glutathione levels in both control and CSE

exposed cells. Importantly, NAC treatment

prevented the CSE-mediated decrease in DICER

protein levels and blocked the shift to higher

molecular mass forms.

We next tested whether the DICER protein

protection seen with NAC led to increased DICER

activity. Using the in vivo activity assay shown in

Figure 3, we transfected a pre-miRNA-146b

expressing vector into Hela cells. Cells were

exposed to CSE with and without NAC (added to

the cell cultures 15 minutes before the CSE).

Figure 7C shows that NAC reverses the loss of

pree-miRNA-146b cleavage seen with CSE

(shown is a representative Northern and

densitometry from three separate experiments).

As increasing cellular glutathione levels leads

to protection from many forms of oxidant stress,

we tested two classic candidate molecular species

commonly associated with cigarette smoke,

hydrogen peroxide (H2O2) and acrolein (a pure

aldehyde compound). As shown in Figure 7D,

supplementing the media of THP-1 cells with

concentrations of H2O2 approaching the limits of

cellular viability did not reproduce the effect of

CSE on immunoreactive DICER levels. In

contrast, acrolein at concentrations within the

range seen in cigarette smoke vapor and in the

epithelial lining fluid of active smokers (37-39)

faithfully reproduced both the decrease in DICER

protein levels and the appearance of higher

molecular mass forms seen after CSE exposure.

DISCUSSION

The major conclusions of the current study

are that cigarette smoke exposure alters miRNA

expression in human alveolar macrophages via

disruption of the complex maturation process that

occurs between miRNA transcription and

generation of the mature transcript. In addition we

demonstrate that the RNA endonuclease DICER,

an essential component of the miRNA biogenesis

pathway, is altered by cigarette smoke exposure.

These conclusions are based on the following

experimental evidence. We show that a group of

miRNAs with significantly decreased expression

in smoker’s cells is not decreased at the

transcriptional level. This suggests a defect in the

processing of the miRNA transcript. We

demonstrate that the lysates of smoke-exposed

cells contain less dsRNA endonuclease activity

with both an in vitro and in vivo assay.

Transfecting in expression plasmids for pre-

miRNAs, we demonstrate that smoke exposure

decreases the conversion of over-expressed pre-

miR146b and pre-miR708 to mature miRNA.

In smoker's alveolar macrophages, we found

alterations in DICER, the RNA endonuclease

responsible for the final processing of the miRNA

stem-loop structure. Exposure to cigarette smoke

led to decreased activity and amounts of the

endonuclease DICER. We demonstrated that

cigarette smoke-exposed cells expressed an

immunoreactive DICER of increased molecular

mass (in reducing conditions). This change in

mass appears to be linked to the addition of

SUMO chains to DICER. Finally, we

demonstrated that the smoking-induced

modifications to intracellular DICER were

blocked by incubation with N-acetylcysteine, were

reproduced by exposure to acrolein, and were less

evident with exposure to H2O2; these data

robustly support a role for aldehydes in cigarette

smoke-induced DICER protein modification and

consequent decrease in miRNA expression.

Collectively, this evidence strongly supports the

concept that alveolar macrophage phenotype in

smoker's lungs is determined, in part, by gene

expression changes driven by a DICER-linked

processing defect in the miRNA system (Figure

7E).

There is increasing evidence that cigarette-

smoking impacts pulmonary pathophysiology

through alterations in miRNA expression in the

lung. We have previously shown that miRNAs are

decreased in alveolar macrophages from smokers

(10). In that paper, we found that smoking down

regulated 1/3 of the tested miRNAs. In addition,

we found a number of correlations between down

regulated miRNAs and up regulated mRNAs,

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Alterations in DICER and miRNA levels with cigarette smoke

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including the emphysema relevant gene, MMP12.

Other studies have supported our discovery of

reduced miRNAs in smoker lung cells. Schembri,

et al, demonstrated differences in miRNA

expression in epithelial cells isolated from the

airways of smokers as compared with cells from

never smokers (40). Similarly, the induced sputum

from active smokers contained a different pattern

of miRNA than the sputum from nonsmokers (41).

In both of these studies, a general decrease in

miRNA expression was noted in the smoking

subjects. No clear differences could be identified

when smokers with and without clinical COPD

were compared, though both groups differed from

nonsmokers (41). Our findings in the alveolar

macrophages retrieved from active smokers show

that miRNA down regulation in response to

cigarette smoke is not restricted to the airway

epithelium. This is in line with the findings of

whole lung miRNA profiles from smoke-exposed

rats (42). Furthermore, our findings that both

THP-1 and Hela cells manifest similar DICER

modifications offer the possibility that cigarette

smoke-induced defects in miRNA processing may

be a more universal cellular response.

These changes in miRNA levels are likely not

simply epiphenomena as all four labs have found

reciprocal changes in mRNA coding genes

relevant to smoking-induced lung disease,

including inflammatory cytokine receptors,

nuclear transcription factors, and

metalloproteinases. In this study we extend our

initial work to demonstrate that the decrease in

miRNAs targeting some of these genes of interest

is not due to a change in miRNA transcription or

intranuclear stability, as there was little difference

in the levels of primary miRNA transcripts

between smokers and nonsmokers despite a

dramatic decrease in mature miRNA strands.

Thus, the smoking-induced decrease in miRNA

levels results from changes in miRNA maturation.

The importance of miRNAs in modulating

health and disease is well accepted. However, how

miRNA levels are modulated remains

incompletely defined. The biogenesis of miRNAs

is complex, but involves a process leading from

nuclear transcription through a final cytosolic

maturational processing. A key step in this

maturation is the cleavage of the stem loop

structure by the cytoplasmic RNA endonuclease,

DICER. Our demonstration that DICER activity is

reduced in smoke-exposed cells led us to focus on

this enzyme. The regulation of DICER appears to

be complex with evidence for autophagy-mediated

protein degradation as well as post-transcriptional

repression of DICER mRNA by the miRNA let-7a

(43). Our lab has previously established that

cigarette smoke induces an autophagic block in

alveolar macrophages and we found no change in

DICER mRNA in smoke exposed cells (9,20).

This led us to postulate that cigarette smoke

must induce some novel change in DICER protein

itself. Immunoblotting of cell lysates demonstrated

the appearance of DICER species in Western gels

(in reducing conditions) of higher molecular mass

suggesting a post-translational protein

modification. This was followed at later times by a

decrease in total DICER mass. By over-expressing

pre-miRNAs, we were able to show a defect in

DICER/complex activity and impaired maturation

at time points when higher molecular weight

DICER species were apparent without notable

change in total DICER mass.

Further analysis of DICER protein provided

evidence for low-level ubiquitination that might

lead to decreased activity or proteasome targeting

of DICER. However, a more significant finding

was an increase in SUMO 2/3 staining of

immunoprecipitated DICER (217 KD). The high

molecular weight species generated by cigarette

smoke exposure were blocked with inhibition of

the dedicated SUMO ligase, Ubc9. SUMO protein

adducts are seen in response to a variety of cellular

stresses including oxidant injury, heat shock, and

DNA strand damage (44-46). DICER has ten

lysine moieties that fulfill described SUMO

consensus sequences (Ψ-K-x-D/E where Ψ is a

hydrophobic residue) (47,48) (Supplement #1).

While Ubc9 may modify many proteins as part of

their respective regulatory processes, this

represents the first description of a change in

DICER function due to post-translational

SUMOylation. Further studies are needed to

dissect whether this may be a potential target for

manipulation of miRNA expression.

Cigarette smoke is a complex potpourri of gas

phase chemicals and particulate biotoxins. These

include potent oxidants and reactive aldehydes

(49,50). We found that smoke-induced

quantitative and qualitative changes in cellular

DICER were abrogated by the concurrent

administration of the potent anti-oxidant and

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Alterations in DICER and miRNA levels with cigarette smoke

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aldehyde-neutralizing agent, NAC. In addition, the

smoke effects on DICER were faithfully

reproduced with acrolein exposure, but not

hydrogen peroxide. Thus, acrolein delivered via

cigarette smoke inhalation might reduce cellular

DICER activity leading to alterations in miRNA

levels and, ultimately, gene expression. Such a

model has been described in cultured endothelial

cells where acrolein exposure resulted in decreases

in miRNA and increased expression of their

respective mRNA targets (39). As acrolein is a

byproduct of the incomplete combustion of many

natural and man-made materials, aldehyde-

induced changes in miRNA expression due to

alterations in DICER activity may prove to be a

universal mechanism of inhalational pollutant

toxicity (37).

The accumulation of alveolar macrophages

in the distal air space is a defining characteristic of

several smoking-associated lung diseases. The

alveolar macrophage is poised as an ideal cell to

reflect the toxic effects of inhaled smoke and

situated to respond in ways that may initiate and/or

perpetuate disease. Cigarette smoke has been

shown to have profound effects on gene

expression profiles of many cells including

alveolar macrophages (5,51-53). The mechanisms

behind this response are complex and likely due to

both genetic and epigenetic responses. This study

describes a novel impact of cigarette-smoke

exposure on the miRNA-processing pathway in

alveolar macrophages. Smoking-induced

alterations in miRNA maturation could

significantly regulate changes in the expression of

disease-relevant genes and represents a new target

for potential therapeutic intervention.

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Acknowledgements. We thank Jiselle Vertinez and Richard Starr for technical assistance.

FOOTNOTES

*This project was supported by NIH R01 HL079901, NIH RO1 HL096625 and R21HL109589 to M. M.

This research was funded in part by the National Institute for Environmental Health Sciences through the

University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH P30 ES005605 and

Grant Number UL1RR024979 from the National Center for Research Resources (NCRR), a part of the

National Institutes of Health (NIH).

1. To whom correspondence should be addressed: Thomas J. Gross, Division of Pulmonary, Critical Care,

and Occupational Medicine, University of Iowa, Iowa City, IA 52242, Phone: (319) 356-6268, Email:

thomas-gross@uiowa.edu 2. Department of Medicine

3. The abbreviations used are: COPD, Chronic Obstructive Pulmonary Disease; CSE, cigarette smoke

extract; SUMO, Small Ubiquitin-like Modifier; siRNA, small interfering RNA; NAC, N-acetyl cysteine

FIGURE LEGENDS

Figure 1. In vivo cigarette smoke exposure alters miRNA profiles in alveolar macrophages. A.

Differentially expressed miRNAs in smoker's alveolar macrophages are down regulated and link to up

regulated mRNAs. Alveolar macrophages were isolated from 4 active smokers (> 10 pack years) and 4

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Alterations in DICER and miRNA levels with cigarette smoke

14

never smokers. An analysis of mRNA expression was performed using Human Exon Array 1.0 ST arrays

from Affymetrix. MiRNA expression was analyzed using TaqMan Low Density Arrays version 2.0

(ABI). Altered miRNAs (> 2 fold change) were analyzed for potential mRNA targets using TargetScan

and TarBase. The miRNA target information was compared to significantly altered mRNAs (q≤ 0.10).

The q value (false discovery rate) for the mRNAs was calculated using the Partek GS implementation of

the step-up method. Gene IDs common to both lists (miRNA targets and mRNAs) were plotted. Each

dot represents a gene/miRNA pair as predicted by Target Scan and/or TarBase. The upper right quadrant

is of particular interest as it identifies down regulated miRNAs that link to up regulated mRNAs. B.

MiRNAs that are down regulated in smoker's alveolar macrophages have intact transcriptional profiles. A

panel of down regulated miRNAs (miR-708, miR-200a, miR-210, miR-187, miR-149, miR-429, miR-

146b-3p, miR-200c) was tested in RNA isolated from smoker and never smoker alveolar macrophages.

Each miRNA was tested using primers specific for the mature miRNA and primers specific for the

primary miRNA transcript (the stem loop structure with accompanying 5' and 3' ends). The data is a

composite of results from 4 smokers and 4 never smokers with significance determined by Student's t test.

Figure 2. Cigarette smoke exposure decreases dsRNA endonuclease activity. To test the effect of

cigarette smoke on DICER activity, we used an in vitro assay of endonuclease cleavage of a fluorescent

27mer target. A. To validate the assay, varying amounts of whole cell lysate protein (THP-1 cells) were

incubated with the FITC-27mer. Shown is a gel demonstrating a dose dependent cleavage of the 27mer

target dsRNA. B. Whole cell lysates from Control THP-1 cells and CSE exposed THP-1 cells (5% CSE

for 6 hours) were incubated with the FITC-27mer. Shown is a fluorescent image of the DNA gel and

densitometry from three separate experiments. Significance was determined using Student's t test. C.

DICER was immunoprecipitated from 500 ug THP-1 protein (control and CSE exposed, 5% for 6 hours).

20% of the resulting protein coated beads (pulled down with an anti-DICER antibody or a nonspecific

IgG) were mixed with the FITC-27mer to assay for DICER-specific ribonuclease activity. Shown is the

fluorescent image of a DNA gel showing both the original 27mer and the cleaved 21mer product.

Densitometry is from three separate experiments.

Figure 3. Cigarette smoke exposure decreases pre-miRNA processing activity. Two highly CSE

regulated miRNAs were chosen for an in vivo pre-miRNA processing. A+B. Transfection with either

pre-miR-146b (panel A) or pre-miR-708 (panel B) and processing of the respective miRNAs was

evaluated by Northern blot and qRT-PCR. Hela cells were transfected with a plasmid expressing pre-

miRNAs under control of the constitutive H1 promoter. 24 hours after transfection, cells were exposed to

1 or 3 hours of CSE and RNA isolated. Northern analysis was performed to demonstrate the relative

degree of pre-miRNA processing. qRT-PCR to compare pre-miRNA to mature miRNA levels was also

performed. All assays were done in triplicate and statistical analysis for both the densitometry data and

qRT-PCR performed (Student's ttest).

Figure 4. DICER protein is modified by cigarette smoke exposure. A. Human alveolar macrophages

freshly isolated from nonsmokers were exposed to varying concentrations of CSE for 6 hours (0.5% to

5.0%). Whole cell lysates were obtained and Western analysis performed for DICER, PACT, TRBP, and

AGO2. Equal loading of the gel was determined by staining for actin. B. Normal human alveolar

macrophages were exposed to CSE (2%) for varying times (1 to 6 hours) Whole cell lysates were

obtained and Western analysis performed for DICER. C. Normal human alveolar macrophages were

exposed to CSE at varying concentrations and whole cell lysates were run on a 7.5 % PAGE SDS gel to

optimize transfer of high molecular weight proteins. PACT protein levels were determined by Western

analysis. D. THP-1 cells were exposed to varying concentrations of CSE for 6 hours; Hela cells were

exposed to CSE for 3 and 6-hour time points. Whole cell lysates were obtained and Western analysis

performed for DICER.

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Alterations in DICER and miRNA levels with cigarette smoke

15

Figure 5. Smoking alters alveolar macrophage DICER protein and not DICER mRNA. A. Human

alveolar macrophages were obtained from active smokers and never smokers. Whole cell lysates were

obtained immediately (baseline sample) and Western analysis was performed for DICER. Shown on the

right is densitometry. Significance was determined using Student's t test. B. Human alveolar macrophages

were obtained from active smokers and never smokers. Fresh whole cell lysates were immediately

analyzed by Western blotting and probed for DICER. The gel was optimized to maximize visualization of

the high molecular weight bands (5% gel with a long transfer). C. Total RNA was obtained from freshly

isolated human alveolar macrophages (43 nonsmoker subjects and 42 smoker subjects). The mRNA levels

for DICER were analyzed using an Affymetrix array (Gene Chip Human Exon Array 1.0 ST arrays).

Shown are the log2 values for DICER, as determined by Partek analysis of the raw data. There is no

difference in DICER mRNA levels between smokers and nonsmokers.

Figure 6. Cigarette smoke exposure increases conjugation of SUMO chains to the protein DICER.

A. Alveolar macrophages from nonsmokers were exposed to 2% CSE in vitro for 6 hours. Whole cell

lysates were isolated and 500 ug of protein was used for immunoprecipitation using an anti-DICER

antibody. The resulting samples were divided into thirds and Western analysis performed for DICER,

SUMO 2/3 and ubiquitin. As a control, 500 ug of whole cell lysate protein was also subjected to

immunoprecipitation with a nonspecific IgG. B. Human alveolar macrophages were exposed to varying

concentrations of CSE. Western analysis was performed for Ubc9. C. Hela cells were incubated with

Ubc9-specific or a control siRNA. The cells were then exposed to 5% CSE for 6 hours and whole cell

protein obtained. Western analysis was performed for DICER, Ubc9 (verifying knockdown) and actin.

Figure 7. DICER modification by cigarette smoke is inhibited by N-acetylcysteine. A. To explore

the chemical properties of the DICER-active component of cigarette smoke, CSE was dialyzed overnight

in tubing with cutoffs of 2 kD or 10 kD. THP-1 cells were then exposed to freshly prepared CSE, CSE

that sat in the refrigerator over night (control for dialysis) or the dialyzed CSE for 6 hours. Western

analysis for DICER was performed on whole cell lysates. B. THP-1 cells were exposed to CSE alone

(2%) or CSE in the presence of NAC (2 mM) for 6 hours. Duplicate assays were set up. One set of cells

was utilized for glutathione assays and the other set of cells were lysed and whole cell protein obtained

for Western analysis for DICER. C. Hela cells were transfected with a plasmid that expresses pre-

miRNA-146b under control of the constitutive H1 promoter. 24 hours following transfection cells were

exposed to CSE (10%) with and without added NAC (2 mM) for three hours. RNA was isolated and

Northern analysis for miRNA-146b performed. Shown is a representative blot and densitometry from

three separate experiments. D. THP-1 cells were exposed to varying concentrations of H2O2 or acrolein

for 6 hours. Whole cell lysates were obtained and Western analysis for DICER performed. E. This

diagram represents an overview of the conclusions supported by the study data.

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Gross et al, 2014

-6

-5

-4

-3

-2

-1

0

1

2

Fold

cha

nge

(sm

oker

s/no

nsm

oker

s)

PrimarymiRs

MaturemiRs

Figure 1

A.

B.

1086420

-2-4-6-8

-10-20 -16 -12 -8 -4 0 4 8 12 16 20

microRNA changes that correlate with target gene expression

RN

A Fo

ld-C

hang

e (S

vs

NS

, q

0.1

0)<

miRNA Fold-Change (S vs NS, Fold 2.0 )<

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Cell lysate (ug)Assay Time (h)

258

108

58

08

Gross et al, 2014

Figure 2

A.

B.

27mer

21mer

Olig

o (D

siR

NA

)

Con

trol

5% C

SE

Con

trol

5% C

SE

IP aDicer IP IgG

C.

Con

trol

CS

E

27mer

21mer

27mer21mer

Non

e

Dic

er A

b

Dic

er A

b

Control CSE

Con

trol I

gG

Con

trol I

gG

Den

sito

met

ry o

f 21m

er(A

rbitr

ary

untis

)

Den

sito

met

ry o

f 21m

er(A

rbitr

ary

num

bers

)

IP antibody

p< 0.01

0

2000

4000

6000

8000

Oligo Con CSE Con CSE0

5001000150020002500300035004000 p<0.01

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Con

trol

No

trans

fect

ion

CS

E 1

h

CS

E 3

h

Con

trol

No

trans

fect

ion

CS

E 1

h

CS

E 3

h

30nt20nt

50nt60nt

20nt

70nt

Pre miR-146b

miR-146b

Pre miR-708

miR-708

Gross et al, 2014

Figure 3.

5’UGAGAACUGAAUUCCAUAGGCUG

3’GG-UCUUGACU-CAGGUGUCCCGU

UGAGCUCUAGCAA

Pre miR-146b (58nt)mature

passenger

Pre miR-708 (68nt)mature

passenger

5’AAGGAGCUUACAAUCUAGCUGGGG

3’GAUCUUCGAGUGUCAGAUCAAC--A

GUAAAUGACUU

GCACAUGAAC

A

Control CSE 1h CSE 3h

Nor

ther

n de

nsito

met

ry, R

atio

(m

atur

e m

iR-1

46b/

pre

miR

-146

b)N

orth

ern

dens

itom

etry

, Rat

io

(mat

ure

miR

-708

/pre

miR

-708

)

miR

-708

RN

A (m

iR-7

08/R

NU

48)

miR

-146

b R

NA

(miR

-146

b/R

NU

48)

A.

B.

Control CSE 1h CSE 3h Control CSE 1h CSE 3h

Control CSE 1h CSE 3h

0

2

4

6

8

10

12

14

****

** p<0.01** p<0.01

**

**

p<0.01*

*

p<0.05 *

*

p<0.05

0

0.1

0.2

0.3

0.4

0.5

0.6

0.000.050.100.150.200.250.300.350.400.45

0

5

10

15

20

25

30

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Gross et al, 2014

Figure 4.

220 kD

mol wtmarker

DICER, 217 kD

Con

trol

CS

E 1

h

CS

E 3

h

CS

E 6

h

hmw DICER

β Actin, 42 kD

A. B.

C. D.

Human alveolar macrophages

Human alveolar macrophages

Con

trol

0.5

% C

SE

2.0

% C

SE

1.0

% C

SE

5.0

% C

SE

PACT, 34 kD

DICER, 217 kD

TRBP, 39 kD

AGO2, 97 kD

hmw DICER

β actin, 42 kD

β actin, 42 kD

Con

trol

2.5%

CS

E

5.0%

CS

E

7.5%

CS

E

10%

CS

E

DICER, 217 kDhmw DICER

THP1 Cells (human acute monocytic leukemia cell line)

Con

trol

CS

E 0

.5%

CS

E 1

.0%

CS

E 2

.0%

CS

E 5

.0%

Hela cells (human cell line derived from cervical cancer)

DICER, 217 kDhmw DICER

β actin, 42 kD

Con

trol

0.5

% C

SE

2.0

% C

SE

1.0

% C

SE

5.0

% C

SE

PACT, 34 kD

hmw PACT

Human alveolar macrophagesβ actin, 42 kD

Con

trol

CS

E 1

0% 3

h

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Gross et al, 2014

Figure 5.

Non

smok

er 1

Non

smok

er 2

Non

smok

er 1

Non

smok

er 2

Non

smok

er 3

Non

smok

er 4

Sm

oker

1

Sm

oker

2

Sm

oker

1

Sm

oker

2

Sm

oker

3

Sm

oker

4

Pk yrs

Pk yrs

0 0 0 0 25 14 12 32

DICER, 217 kD

β Actin, 42 kD

220

mw

0

500

1000

1500

2000

2500

3000

3500

4000

Nonsmokers Smokers

Den

sito

met

ry (a

rbitr

ary

units

)

p<0.05

DICER, 217 kD

hmw DICER

β Actin, 42 kD

A.

B. C.

Human alveolar macrophages

0 0 10 20

Human alveolar macrophages Nonsmokers (n=43) Smokers (n=42

Dic

er m

RN

A (lo

g2 v

alue

s)9.0

8.5

8.0

7.5

7.0

6.5

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Gross et al, 2014

Figure 6.

B.

C.

A.

Con CSE Con CSE Con CSE Con CSE

anti-Dicer

Ubc9, 18kD

anti-Sumo 2/3 anti-Ub

Pull down

DICER, 217 kD

DICER Ab DICER Ab DICER AbControl IgG

hmw DICER

Western Ab

DICER, 217 kD

Ubc9, 18 kD

siRNA amts

Ubc9 siR

NA

con siR

NA

CSE + Ubc9 siR

NA

CSE + con siR

NA

hmw DICER

β actin, 42 kD

β actin, 42 kD

Con

trol

0.5

% C

SE

2.0

% C

SE

1.0

% C

SE

Human alveolar macrophages

Human alveolar macrophages

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Con

trol

5% fr

esh

CS

E

5% 2

K d

ialy

zed

CS

E

5% 1

0K d

ialy

zed

CS

E

5% C

SE

(4o

over

nigh

t)DICER 217 kD

hmw DICER

β actin, 42 kD

β actin, 42 kD

β actin, 42 kD

DICER, 217 kD

Con

trol

NA

C

CS

E

CS

E +

NA

C

hmw DICER

β actin, 42 kD

Gross et al, 2014

Figure 7

A.

B.

C.

D. E.

Con

trol

5% C

SE

50 u

M a

crol

ein

100

uM a

crol

ein

150

uM a

crol

ein

250

uM a

crol

ein

Con

trol

5% C

SE

100

uM H

2O2

250

uM H

2O2

500

uM H

2O2

1 m

M H

2O2

DICER, 217 kDhmw DICER

DICER, 217 kDhmw DICER

0

400

800

1200

1600

2000

ControlNACCSE

NAC + CSE

Glu

tath

ione

(uM

)

Cigarette smoke

Aldehydes/oxidants

Dicer modification

Large scale decreasein mature microRNAs

Increase in selected gene expression

MicroRNA processing defect

Con

trol

No

trans

fect

ion

CS

E 3

h

NA

C+C

SE

NA

C

Pre miR-146b

miR-146b

Control

Den

sito

met

ryR

atio

(miR

-146

b/P

re m

iR-1

46b)

NAC CSE Both0.00

0.05

0.10

0.15

0.20

0.25

*

p<0.05

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Khushboo Goel, Alicia K. Gerke, Ihab H. Hassan and Martha M. MonickThomas J. Gross, Linda S. Powers, Ryan L. Boudreau, Brandi Brink, Anna Reisetter,

the endonuclease DICERA microRNA processing defect in smokers' macrophages is linked to SUMOylation of

published online March 25, 2014J. Biol. Chem. 

  10.1074/jbc.M114.565473Access the most updated version of this article at doi:

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