Title: SRF Mediates Differentiation and Activation of Myofibroblasts in Pulmonary Fibrosis
1SRF Mediates Differentiation and Activation of
Myofibroblasts in Pulmonary Fibrosis
2Hypothesis
Stated early
- Serum Response Factor (SRF), a transcription
factor involved in smooth muscle myogenesis, is
necessary for the differentiation of fibroblasts
into myofibroblasts during fibrosis.
3The Importance?
Of problem
- Idiopathic Pulmonary Fibrosis (IPF)
- - devastating disease with 70 mortality.
- Lesions in lungs
- - damaged aveolar epithelial cells in lungs
promote fibroblast proliferation, including an
unique form, myofibroblasts. - - myofibroblasts promote extracellular matrix
deposition, stiffening of interstitial tissue,
and an inflammatory response.
http//noairtogo.tripod.com/ipf-pics.htm
4Introduction - Myofibroblasts
What you need to know the players
- Unique form of fibroblast.
- Contains myofilaments and smooth muscle (SM)
a-actin, and produces high level of collagen. - Present only during wound healing or in fibrosing
diseases secretes proapoptotic factor.
5Introduction Model System
- The model system - bleomycin-pulmonary induced
fibrosis in mice. - Mimics injury pattern in IPF, but limitations.
- Anti-cancer drug induce free radicals.
Grande et al. 1998. Lung Fibrosis induced by
bleomycin. Scan. Mic. 12487-495.
6Introduction TGF-ß1
- Transforming Growth Factor ß1 (TGF- ß1) is one of
several cytokines found elevated in IPF. - Critical mediator of lung fibrosis
- - Overexpression induces fibrosis in vivo.
- - Treatment with agonist prevents fibrosis in
vivo. - - Knockout of downstream signal of TGF-ß1
- receptor eliminates fibrosis.
7Introduction TGF-ß1 Myofibroblasts
- Association between TGF-ß1 myofibroblasts
- - TGF-ß1 induces differentiation of lung
fibroblast culture into myofibroblasts in vitro. - - In situ hybridization shows myofibroblasts
- as a significant source of TGF-ß1.
8Introduction Serum Response Factor (SRF)
- SRF - member of MADS box family.
- Regulates embryonic development and a variety of
muscle-specific genes (SM a-actin).
9Introduction - SRF Fibrosis
- Yang et al. (2003) found
- - Elevated SRF levels in bleomycin induced
fibrosis. - - In vitro overexpression of SRF showed
upregulation of several genes involved in
myofibroblast activation (TGF-ß1, SM a-actin). - Yang, Y., Zhe, M., Phan, S., Ullenbruch, M., and
Schuger, L. 2003. Involvement of SRF isoforms
in myofibroblast differentiation during
bleomycin-induced lung injury. Am. J. Respir.
Cell Mol. Biol. 29 583-590.
10Proposed Work
- To examine the effect that changes in SRF levels
will have on fibrosis both in vitro and in vivo. - Significance
- - Myofibroblast proliferation/activation is
believed essential for IPF development. - - Through manipulation of SRF, myofibroblast
function could be altered and demonstrate a
proliferative or inhibitory effect on lung
fibrosis.
Of proposed work
11Specific Aim 1
What do I want to do?
- To test if the transcription factor SRF is
essential for fibroblast differentiation after
trying to induce differentiation with TGF-ß1 in
vitro.
12Specific Aim 1 Rationale
Why do it?
- SRF involved in smooth muscle myogenesis.
- Yang et al. (2003) showed elevated levels of SRF
during fibrosis and elevated expression of genes
involved in myofibroblast activation. - - Suggests SRF could play a role in the
- differentiation of fibroblasts into
myofibroblasts. - - Can test in vitro using TGF-ß1 to induce
- fibroblast differentiation.
13Specific Aim 1 Design
How will I do it?
- Simple in vitro model tested first
- - Antisense oligos to SRF will be added to mouse
fibroblast culture to suppress, check with
RT-PCR. - - TGF-ß1 will be added to induce
differentiation. - Results will be visualized with
immunofluorescence staining - - Antibodies to SM a-actin and collagen I act as
specific markers to myofibroblast
differentiation. - - Compare to negative (TGF-ß1 -) /positive
controls (TGF-ß1, antisense -).
14Specific Aim 1 Results
- Help establish if fibroblasts are sensitive to
TGF-ß1 for myofibroblast differentiation after
SRF is knocked down. - If blocked, could have significant impact on
development of lung fibrosis. - If no change, could mean SRF has no role, but in
vitro system very limited.
What will results mean?
15Specific Aim 2
- To determine if overexpression of SRF in
fibroblasts will promote fibroblast
differentiation and/or activation in vivo.
16Specific Aim 2 Rationale
- Similar rationale to Specific Aim 1 regarding
Yang et al. (2003) experiments. - In vivo experiments using well-established
bleomycin induced-fibrosis model could provide
more complete picture - - Myofibroblast differentiation known to be
affected by more than just TGF-ß1.
17Specific Aim 2 Design
- Want SRF overexpression to be specific to
fibroblasts and only expressed after
embryogenesis complete. - Fibroblast specific protein (FSP1) is
specifically expressed in fibroblasts so promoter
can be used to target. - FSP1 function unknown cell motility?
18SA 2 Design Continued
- To ensure proper development, must delay SRF
overexpression use variation of
ligand-inducible binary transgenic system.
Kwak, et al. 2004. Ann. Rev. Physiol.
66647-663.
- In absence of ligand, regulator
- (black) is inactive, and gene of interest
- (GOI) is not expressed.
- Presence of the ligand (pentagon)
- converts the regulator into an active
- form.
- The activated regulator binds to
- response element and activates
- transcription of GOI (4).
19SA 2 Design Continued
- The gene of interest (GOI) to be overexpressed is
placed under control of the tet operon (tetO) -
tetracycline resistance operon of E.coli. - A second construct contains the positive
regulator, the reverse tet transactivator (rtTA) - - Can only activate tetO after the ligand binds
to the regulator rtTA. Ligand is a homolog of
tetracycline doxycycline (Dox). - - Second construct is placed under
transcriptional control of the tissue-specific
promoter (FSP1).
20SA 2 Design Continued
Dox
- An additional step not shown in the diagram is
done to prevent leaky expression.
rtTA under control of FSP1 promoter
tetO
SRF
- Third construct is present containing a silencer
tTS - - Binds to tetO and prevents leaky expression.
- Transcriptional repressor that is inactivated by
the presence - of the ligand, Dox.
21SA 2 Design Continued
- Constructs readily available except the
fibroblast/SRF specifics must be subcloned in. - Constructs pronuclear microinjected into mice
eggs. - - Check success through tail biopsies of mice
using Southern analysis and PCR. - - Test large number at once (low success rate).
22SA 2 Design Continued
- Induce pulmonary fibrosis in 6 week old
transgenic mice through intratracheal injection
0.15 U/kg of bleomycin (and have saline-only
control). - Induce SRF overexpression through feeding mice
0.5 mg/ml Dox (plus water-only as control). - Sacrifice mice at 1, 3, 7, 14, and 21 days for
analysis to compare severity of fibrosis to
controls.
23SA 2 Design Assays
- Indicators of fibrosis
- Collagen levels measure via Sircol kit
(elongated dye). - TGF-ß1 levels measure via quantitative RT-PCR
(GAPDH normalization) after isolate total RNA
from lung tissue (Trizol reagent). - General morphology fix slices of lung tissue
obtained via cryostat. - - Examine lesions.
- - Look for myofibroblasts (SM a-actin Ab,
- collagen stained with picosirius red).
24SA 2 Assays Cont
- Myofibroblast cell count
- Fluorescence Activated Cell Sorter (FACS) use
flow cytometry with fluorescent probes. - - Take numerous cell samples from lungs to get
representative count. - Label collagen and SM a-actin with antibodies
conjugated to different fluorescing dyes.
25Specific Aim 2 Results
- If SRF is a key mediator of myofibroblast
differentiation/activation, overexpression would
likely induce a more severe case of fibrosis. - - Severity will be measured with collagen/TGF-ß1
levels and morphology. - - Number of myofibroblasts (FACs) will be
compared to myofibroblast activity levels
(indicator assays).
26SA 2 Results Cont
- Instead of final fibrotic state being more
severe, may change rate. - Potential of no observable change, then move to
Aim 3. - - Prior experiments (overexpression of SRF in
heart, overexpression of TGF-ß1 in lungs) suggest
likely result.
27Specific Aim 3
- To test if conditional deletion of SRF within
fibroblasts will affect myofibroblast
differentiation and the progression of lung
fibrosis in vivo.
28Specific Aim 3 Rationale
- Conditional deletion of SRF could help answer if
myofibroblasts are necessary for the onset of
pulmonary fibrosis. - At the very least, conditional deletion will
examine any changes in fibrosis due to the
deletion within fibroblasts, including
dysregulation of TGF-ß1 expression.
29Specific Aim 3 Design
- The design/assays are near identical to Specific
Aim 2. - Proper embryonic development requires SRF, so
conditional deletion - - Again, use tetracycline transactivator system
except now inducing gene ablation. - - Induce ablation with Cre-loxP recombinase.
30SA 3 Design Continued
http//www.ruf.rice.edu/rur/issue1_files/norman.h
tml
Insert LoxP sites around gene of interest, SRF,
through homologous recombination in embryonic
stem cells. Have Cre under control of TetO
response element, and induce production of Cre
through adding Dox.
31Specific Aim 3 Results
- If SRF necessary for myofibroblast
differentiation, deletion should mostly eliminate
their presence. - - Collagen I levels would drop.
- - SM a-actin would be restricted to smooth
muscle. - - Fibrosis would potentially be eliminated or
significantly reduced. - If SRF found necessary for differentiation,
deletion could have a milder effect on fibrosis. - - Suggests SRF still necessary for full onset.
- - Non-differentiated fibroblasts compensate?
32SA 3 Results Cont
- If SRF not found necessary for differentiation,
still can see how deletion affects fibrosis. - - Potential dysregulation of transcriptional
control over TGF-ß1, SM a-action expression, etc. - - Slower to reach peak fibrosis.
33Future Work
- If SRF found necessary, examine any differences
in expression/regulation between mesenchymal
fibroblasts vs. blood circulating fibroblasts. - Look for any co-factors associated with SRF
involved in myofibroblast regulation.