SRF Mediates Differentiation and Activation of Myofibroblasts

in Pulmonary Fibrosis

Jeff Crawford

Hypothesis

Serum Response Factor (SRF), a transcription factor involved in smooth muscle myogenesis, is necessary for the differentiation of fibroblasts into myofibroblasts during fibrosis.

Stated early

The Importance?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

Of problem

Introduction - MyofibroblastsUnique form of fibroblast.

Contains myofilaments and smooth muscle (SM) α-actin, and produces high level of collagen.

Present only during wound healing or in fibrosing diseases – secretes proapoptotic factor.

What you need to know;the “players”

Introduction – Model SystemThe 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. 12:487-495.

Introduction – TGF-β1

Transforming Growth Factor β1 (TGF- β1) 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-β1 receptor eliminates fibrosis.

Introduction – TGF-β1 & Myofibroblasts

Association between TGF-β1 & myofibroblasts:- TGF-β1 induces differentiation of lung fibroblast

culture into myofibroblasts in vitro.

- In situ hybridization shows myofibroblasts as a significant source of TGF-β1.

Introduction – Serum Response Factor (SRF)

SRF - member of MADS box family.

Regulates embryonic development and a variety of muscle-specific genes (SM α-actin).

Introduction - SRF & FibrosisYang 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-β1, SM α-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.

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

Specific Aim 1

To test if the transcription factor SRF is essential for fibroblast differentiation after trying to induce differentiation with TGF-β1 in vitro.

What do I wantto do?

Specific Aim 1 RationaleSRF 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-β1 to induce fibroblast differentiation.

Why do it?

Specific Aim 1 DesignSimple in vitro model tested first:- Antisense oligos to SRF will be added to mouse

fibroblast culture to suppress, check with RT-PCR.

- TGF-β1 will be added to induce differentiation.

Results will be visualized with immunofluorescence staining:

- Antibodies to SM α-actin and collagen I act as specific markers to myofibroblast differentiation.

- Compare to negative (TGF-β1 -) /positive controls (TGF-β1+, antisense -).

How will Ido it?

Specific Aim 1 ResultsHelp establish if fibroblasts are sensitive to TGF-

β1 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 resultsmean?

Specific Aim 2

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

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-β1.

Specific Aim 2 DesignWant 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?

SA 2 Design Continued

To ensure proper development, must delay SRF overexpression – use variation of ligand-inducible binary transgenic system.

1. In absence of ligand, regulator(black) is inactive, and gene of interest(GOI) is not expressed.

2. Presence of the ligand (pentagon)converts the regulator into an active form.

3. The activated regulator binds to response element and activatestranscription of GOI (4).

Kwak, et al. 2004. Ann. Rev. Physiol. 66:647-663.

SA 2 Design ContinuedThe 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).

SA 2 Design Continued

An additional step not shown in the diagram is done to prevent leaky expression.

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.

rtTAunder control of FSP1 promoter

tetO SRF

Dox

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).

SA 2 Design ContinuedInduce 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.

SA 2 Design AssaysIndicators of fibrosis:Collagen levels – measure via Sircol kit (elongated

dye).

TGF-β1 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 α-actin Ab, collagen stained with picosirius red).

SA 2 Assays Con’t

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 α-actin with antibodies conjugated to different fluorescing dyes.

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-β1 levels and morphology.

- Number of myofibroblasts (FACs) will be compared to myofibroblast activity levels (indicator assays).

SA 2 Results Con’t

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-β1 in lungs) suggest likely result.

Specific Aim 3

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

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-β1 expression.

Specific Aim 3 DesignThe 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.

SA 3 Design Continuedhttp://www.ruf.rice.edu/~rur/issue1_files/norman.html

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.

Specific Aim 3 ResultsIf SRF necessary for myofibroblast differentiation,

deletion should mostly eliminate their presence.- Collagen I levels would drop.

- SM α-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?

SA 3 Results Con’t

If SRF not found necessary for differentiation, still can see how deletion affects fibrosis.

- Potential dysregulation of transcriptional control over TGF-β1, SM α-action expression, etc.

- Slower to reach peak fibrosis.

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.