SRF Mediates Differentiation and Activation of Myofibroblasts in Pulmonary Fibrosis

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SRF Mediates Differentiation and Activation of
Myofibroblasts in Pulmonary Fibrosis

• Jeff Crawford

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Hypothesis
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.

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The 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
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Introduction - 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.

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Introduction 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.
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Introduction 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.

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Introduction 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.

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Introduction Serum Response Factor (SRF)

• SRF - member of MADS box family.
• Regulates embryonic development and a variety of
  muscle-specific genes (SM a-actin).

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Introduction - 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.

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Proposed 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
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Specific 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.

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Specific 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.

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Specific 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 -).

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Specific 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?
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Specific Aim 2

• To determine if overexpression of SRF in
  fibroblasts will promote fibroblast
  differentiation and/or activation in vivo.

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Specific 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.

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Specific 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?

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SA 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).

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SA 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).

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SA 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.

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SA 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).

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SA 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.

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SA 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).

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SA 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.

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Specific 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).

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SA 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.

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Specific Aim 3

• To test if conditional deletion of SRF within
  fibroblasts will affect myofibroblast
  differentiation and the progression of lung
  fibrosis in vivo.

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Specific 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.

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Specific 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.

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SA 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.
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Specific 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?

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SA 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.

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Future 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.