Cardiac hypertrophy - Molecules and drugs involved
Roundtable 6-8-13mv1-3-3
1. The Induction of PU.1 in IL-1-induced Myeloid
Differentiation
Gena V. Topper
Passegué Lab
Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF
University of California, San Francisco
Roundtable 8/6/13
2. Granulocyte/
Macrophage
Progenitor (GMP)
Multipotent progenitor (MPP)
Common
Lymphoid
Progenitor (CLP)
Hematopoietic Stem Cell (HSC)
RBC
Meg
Platelets
Neu Eo Bas
Mon
Mac
T cell B cell NK cell
Common Myeloid
Progenitor (CMP)
Megakaryocyte/
Erythrocyte
Progenitor (MEP)
Hematopoiesis is the process of blood generation from HSCs
Specialization
Self-renewal
• Quiescent
• Generates all blood lineages
• High self-renewal capacity
• Compartment of amplification
• Very proliferative
• Progressively more defined
lineage potential
• Terminally differentiated
• Most specialized and common,
bulk of blood system
3. Current and Emerging Views of Myelopoiesis
• Classical view: a process controlled by sequential activation of lineage-specific
transcription factors
• This view ignores the potential of the microenvironment and/or systemic cytokine
environment to influence the outcome/direction of HSC differentiation
• Can cell-extrinsic factors alter HSCs fate choices to facilitate myeloid
differentiation?
HSC
PU.1
PU.1
PU.1PU.1, NF-κB, C/EBPα
4. The Role of IL-1 in Myelopoiesis
• IL-1 is a protypical proinflammatory cytokine known to regulate myelopoiesis
• IL-1 therapy given to sublethally irradiated mice resulted in faster recovery
of the myeloid compartment, but a smaller number of pre-B and lymphoid
cells (Morrissey, et al. 1988)
• IL-1 known to drive expansion of myeloid cells in BM progenitors through
autocrine GM-CSF signaling (Bot, et al. 1990)
• HSC and MPP proliferation in response to alum-induced inflammation has
been shown to be IL-1R-dependent (Ueda, et al. 2009)
• IL-1 is an extrinsic signal that promotes myelopoiesis, but by what
mechanism?
7. PU.1 is upregulated by IL-1β in HSCs
Is IL-1 altering
expression of myeloid
lineage determinants?
12h Liquid culture +/- IL-1β
0
0.5
1
1.5
2
2.5
Pu.1
Expression(Log2)vs.-IL-1β
C/ebpα
HSCs
24 hrs
Analysis
+/- IL-1β
eYFPPu.1
Pu.1-EYFP expression in HSCs
24hrsinculture+IL-1β
-IL-1β
+IL-1β
FITC, Living Cells
Methocult
8. PU.1 is a Potent Transcription Factor
• Transcription factor that mediates
myeloid and B cell differentiation
• Pu.1 expression increases as HSCs
differentiate along the myeloid axis
• PU.1 drives a molecular program
characterized by the induction of
myeloid determinants such as CD18,
CSFRs, Mac1, etc
• Pu.1 hypomorphs show impaired
myeloid differentiation
Friedman AD (2007), Oncogene
14. Conclusions
• Extrinsic signals are capable of altering HSC fate
• IL-1 directly activates a GM differentiation program in HSCs characterized
by increased expression of PU.1 and its downstream target genes
• Restricted to only the most immature HSC compartments as GMPS are not
responsive to Il-1 in this way
HSC
IL-1
IL-1RI
M-CSFR
Mac-
1
FCgR
GM-
CSFR
Pu.1 Pu.1 Division Division
GM-“primed” HSC
GM Progenitors GM
Cells
15. Future Questions
1. To what extent does IL-1 drive PU.1-dependent differentiation in
intermediate progenitor compartments?
2. Does IL-1 drive myeloid differentiation through increased expression of
cytokines such as M-CSF and GM-SCF in an autocrine loop?
16. Emmanuelle Passegué
Eric Pietras
Matt Warr
Stephanie Leong
Johanna Flach
Sietske Bakker
Latika Kohli
Silvia Alvarez
Cristina Mirantes-Barbeito
Acknowledgements
Thank you all for a fantastic
educational summer!!!
Have a wonderful 2013
At the top we have the typical HSC - self-renewing, quiescent, low metabolic activity, responsible for the production of all blood cells
HSCs are unique given their self-renewal capacity and differentiate into progenitors
Next is the compartment of amplification, which is important because HSCs are so rare
This is where the more-differentiated progeny of the HSC reside – higher metabolic activity, more restricted lineage, generate only specific fully differentiated cells
Which brings us to the compartment of cells that are fully specialized and differentiated, some of them unable to even replicate themselves, bulk of system
Project mostly deals with myeloid differentiation
Stochastic activation of specific TFs that leads to differentiation of HSCs to cell of myeloid lineage
All HSCs have low levels of Pu.1, C/EBPa, etc amounts increase as they differentiate
When HSCs differentiate, we see ^ of these factors increase in expression, thought to be primary mediators --> affected by extrinsic sources such as autocrine/niche signaling?
“one extrinsic factor that our lab has been investigating is IL-1, and previous studies have laid a solid groundwork of evidence for its study”
Inflammatory cytokine known to drive myelopoiesis, extrinsic signals actually have a big and important effect
“Previously, Jose Marc performed this experiment by culturing HSCs […] to observe the effects of colony formation and content with and without IL-1b”
Amount of ‘mix’ colonies is a representation in immaturity so less ‘lineage’ choices have been made for each cells nothing instructed the cell in a certain way (colonies are large and multilobed)
IL-1b colonies have tightly-packed morphology, very pure, cells have been pushed to GM lineage
“Clearly Il-1b pushes HSC to a mature myeloid lineage faster then without”
We don’t know how IL-1 does this, so this is the question we are investigating
“as a complimentary approach, we used a liquid culture assay to show the evolution of surface markers over time”
consistent with previous results
“this striking alteration of HSC surface markers showing the accelerated acquisition of mature markers and less of immature markers, showing that IL-1 is driving accelerated myeloid differentiation”
“we first decided to look at how IL-1 was regulating the transcription of important myelopoiesis factors using qRT, and we found that Il-1 specifically upregulated PU.1 in HSCs, whereas we saw no specific effect for another TF, C/EBPa in HSCs”
“in addition to this, we validated this data using the PU.1 reporter mouse”
so what is PU.1?
TALK ABOUT THE FIGURE!
Saw Pu.1 ^ so looked at other myeloid determinants
First graph are direct targets
Second graph with genes shown to be downregulated without PU.1, other cytokines that drive myelopoiesis
“this is evidence that Il-1 activates a specific PU.1-driven molecular program in HSCs, so does this happen in more differentiated myeloid cells?
“If we look at the MFI, the brightness of the Il-1 samples is consistently higher, but they converge around day 6 as the effect of IL-1 lessens as the cells mature”
“IL-1 is clearly inducing PU.1, but the induction is sustained only for awhile, leading us to the question of whether Il-1-induced PU.1 expression affects cells they are already somewhat differentiated”
Pu.1 expression in myeloid progenitors is not driven by IL-1 signaling once the cell is already committed to myeloid differentiation
No change at all in GMP as the effect of PU.1 is only a property of cells uncommitted to myeloid lineage
High expression but no CHANGE/no PU.1 INDUCTION
Values for ctrl AND il1 remain stable throughout experiment so far
Il-1 doesn’t affect differentiated progenitors, and effect gets increasingly smaller along the axis
demonstrated by IL-1 influencing and accelerating myeloid differentiation, by regulating specific transcription factors within the network such as PU.1, activating a specific molecular program
The Sieweke lab (Nature, April 2013) was able to show that PU.1 expression is induced by M-CSF through a very potent upregulatory circuit
we see increased expression of myelopoietic cytokines in our qRT-PCR data
We could validate this by quantifying cytokine output by HSCs from regular and M-CSFR knock-out mice with and without IL-1 during myeloid differentiation in vitro
