Coordinated Regulation of Extracellular Matrix
Synthesis by the MicroRNA-29 Family in the
Guadalupe Villarreal, Jr, Dong-Jin Oh, Min Hyung Kang, and Douglas J. Rhee
PURPOSE. The microRNA-29 (miR-29) family has emerged, in
various tissues, as a key modulator of extracellular matrix
(ECM) homeostasis. In this study, the authors investigate the
role of the miR-29 family in the regulation of ECM synthesis in
the trabecular meshwork (TM) under basal and TGF-?2 stimu-
METHODS. Human TM cells were incubated with 2.5 ng/mL
activated, recombinant human TGF-?2 for 24, 48, and 72
hours. A specific pharmacologic inhibitor was used to block
SMAD3 function in the context of TGF-?2 stimulation. Changes
in the expression of the miR-29 family were assessed by real-
time PCR. The effect of miR-29 molecules and inhibitors on
ECM levels was determined by immunoblot analysis.
RESULTS. All three members of the miR-29 family were ex-
pressed in cultured TM cells. Although the incubation of TM
cells with TGF-?2 induced miR-29a and suppressed miR-29b
levels, no significant effect was observed on miR-29c expres-
sion. Additional studies revealed that SMAD3 modulates miR-
29b expression under basal and TGF-?2 conditions. Subse-
quent gain- and loss-of-function experiments demonstrated that
the miR-29 family functions as a critical suppressor of various
ECM proteins under basal and TGF-?2 stimulatory conditions.
CONCLUSIONS. The findings derived from this study identify
the miR-29 family as a critical regulator of ECM expression in
the TM and suggest that its modulation by TGF-?2 may be
important in controlling ECM synthesis. Together, these data
provide further insight into the complex regulatory mecha-
nisms mediating TGF-?2 signaling and ECM production in
the TM. (Invest Ophthalmol Vis Sci. 2011;52:3391–3397)
causes of blindness worldwide.1,2In the United States, glau-
coma is the leading cause of preventable blindness in persons
of Latino or African descent.3,4Despite modern treatment, the
estimated risk of blindness from glaucoma over a 15- to 20-year
period is between 7% and 24%.5,6Even in patients with im-
paired vision, but not blindness, vision loss can have profound
rimary open-angle glaucoma (POAG) is a chronic, degen-
erative optic neuropathy that represents one of the leading
consequences on daily functioning. For example, the extent of
visual field loss from glaucoma correlates to both an increased
risk of a motor vehicle accident and a fall with injury.7,8Recent
studies suggest that the number of Americans with open-angle
glaucoma will increase by 50% to 4.2 million by the year 2020.9
The progressive optic nerve damage seen in glaucoma re-
sults from an elevation of intraocular pressure (IOP) beyond
the structural and vascular capabilities of the optic nerve to
withstand. IOP is determined by the balance between aqueous
inflow and outflow. In POAG, elevated IOP results from im-
paired aqueous outflow.10Normally, between 80% and 90% of
aqueous outflow occurs through the trabecular meshwork
(TM), with the remaining 10% to 20% occurring through the
ciliary body face.11,12The TM is the critical tissue for the
regulation of aqueous outflow, and its dysregulation causes
elevated IOP.13The tissue that includes the inner wall of
Schlemm’s canal and the juxtacanalicular (JCT) region of the
TM is the anatomic location of highest outflow resistance.14
The JCT region is an amorphous and dynamic layer composed
of endothelial cells and extracellular matrix (ECM). Alterations
in the balance between ECM synthesis and breakdown influ-
ence aqueous outflow.15–19Hence, a rigorous understanding of
the molecular mechanisms involved in the control of ECM
homeostasis is pivotal for the development of new therapies
The transforming growth factor-beta (TGF-?) family of cy-
tokines functions as an important regulator of wound healing
and ECM synthesis.20One such isoform, TGF-?2, has been
shown in several studies to be elevated in the aqueous humor
of POAG patients.21–24Tripathi et al.24report total aqueous
humor TGF-?2 levels (mean ? SD) to be 1.48 ? 0.68 ng/mL in
healthy human subjects compared with 2.70 ? 0.76 ng/mL in
age-matched POAG patients. In addition, the authors show that
aqueous humor levels of intrinsically active TGF-?2 were ele-
vated in POAG patients (0.45 ? 0.28 ng/mL) compared with
age-matched healthy subjects (0.20 ? 0.24 ng/mL).24Subse-
quent work has since demonstrated that TGF-?2 decreases
aqueous humor outflow facility, which, in turn, elevates
IOP.20,25,26One possible mechanism for the decrease in out-
flow facility may involve the associated TGF-?2–mediated in-
crease in ECM accumulation in the TM.20,25,26Specifically,
TGF-?2 has been reported to stimulate the expression of fi-
bronectin, thrombospondin-1, laminin, collagen I, and collagen
IV, key ECM components in the TM.26–29
MicroRNAs are small (?22 nucleotides), single-stranded
RNAs that modulate the posttranscriptional expression of
genes.30These short RNAs are predicted to regulate as many as
30% of human mRNA transcripts, with a given microRNA
potentially binding up to 200 gene targets.31Recent studies
have suggested a critical involvement of microRNAs in the
development of tissue fibrosis, with the microRNA-29 (miR-29)
family serving as an important mediator of these effects.32–34In
nonocular human tissues, the miR-29 family regulates a number
From the Department of Ophthalmology, Massachusetts Eye & Ear
Infirmary, Harvard Medical School, Boston, Massachusetts.
Supported by a Fight for Sight Summer Student Fellowship (GV)
and by National Eye Institute Grants EY 019654-01 (DJR) and EY
014104 (MEEI Vision-Core Grant).
Submitted for publication July 5, 2010; revised January 3, 2011;
accepted January 19, 2011.
Disclosure: G. Villarreal, Jr, None; D.-J. Oh, None; M.H. Kang,
None; D.J. Rhee, None
Corresponding author: Douglas J. Rhee, Department of Ophthal-
mology, Massachusetts Eye & Ear Infirmary, 243 Charles Street, Boston,
MA 02114; firstname.lastname@example.org.
Investigative Ophthalmology & Visual Science, May 2011, Vol. 52, No. 6
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
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