role of gp130 activation for hsc self-renewal

Role of gp130 Activation for HSC Self-renewal

Aurore Degrange and Lisa Treat

BE.400 Term ProjectDecember 10, 2002

Brief Overview

BackgroundGoalsPrevious experimental resultsComputational modelProposed experimentsConclusions

Hematopoietic Stem Cell (HSC) Fate

www.nih.gov/news/stemcells/chapter5.pdf

Background

Cytokines: soluble proteins HSC proliferation HSC differentiation

Some signal through common subunits LIF: LIFR + gp130 IL-6: IL-6R + gp130

HIL-6

Fusion of IL-6 and soluble IL-6 receptorFully active at 100- to 1000-fold lower concentrations compared to unlinked

Peters et al. (1998)

Goals

Isolate impact of gp130 activation in promoting HSC self-renewalAdapt ESC computational model by Viswanathan et al. for HSC systemDesign experiments To determine unknown parameters To validate the model

Important Role of gp130 Activation in HSC Self-renewal

Fixed concentrations of SCF and FLFor HIL-6, narrow range of effective concentrations Independent of particular ligand used to stimulate gp130 receptor

Adapted from Audet et al. (2001)

Self-r

en

ew

ing H

SC

Popula

tion

Expansi

on (

Fold

)

Model System of gp130 Activation

LIF

LIF LIF

LIFR

LIFR

LIFR

gp1

30

gp1

30

KC1

KD1

gp1

30

gp1

30

KC2KD2

gp1

30

gp1

30

HIL-6

HIL-6

HIL-6

Activation by LIFKD1 = LIF dissociation rate

constant

KC1 = cross-linking rate constant

Activation by HIL-6KD2 = HIL-6 dissociation rate

constant

KC2 = cross-linking rate constant

Adapted from Viswanathan et al. (2002)

Activation by LIFLIF

LIF LIF

LIFR

LIFR

LIFR

gp1

30

gp1

30

KC1

KD1


111 CRL DK

LIFK CRC C 1

21

1

11 C

LRKD

At steady-state:

LIFC C

RCK 21

1

2

12

2

1111

θ4χαα1χαα1

2

1T

CD

LIFLIF

CD

LIFLIFLIF RL

KKKKLC

where:

LIFT

LIFT

CRR

CCRR

22

111

211

1

χ

α

TTC

DLIF

RRK

LK

2

1θT

T

R

R

Activation by HIL-6


122 CRL DK

6212

HILK CRC C

1

22 C

LRKD

At steady-state:

6

212

HIL

C C

RCK

2

1γα8α1α1

α8

α16

266

26

66

HILHILHIL

CHIL

HILHIL KC

where:

6122 2 HILT CCRR LKDHIL 26α 22γ TC RKgp1

30

gp1

30

KC2KD2

gp1

30

gp1

30

HIL-6

HIL-6

HIL-6

10-13

10-12

10-11

10-10

10-9

10-8

10-7

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1x 10

-10

LIFHIL-6

Modeled gp130 Activation by LIF & HIL-6

Sig

nalin

g C

om

ple

x N

um

ber

(M)

Ligand Concentration (M)

10

-1310

-1210

-1110

-1010

-910

-810

-70

0.2

0.4

0.6

0.8

1

1.2x 10

-10

10-13

10-12

10-11

10-10

10-9

10-8

10-7

0

0.2

0.4

0.6

0.8

1

1.2x 10

-1010

-1310

-1210

-1110

-1010

-910

-810

-70

0.5

1

1.5

2

2.5x 10

-10

Effects of Varying Parameters on CLIF

Increasing LIF receptor numbers

Increasing gp130 receptor numbers

LIF Concentration (M)

Sig

nalin

g C

om

ple

x N

um

ber

(M)

10-13

10-12

10-11

10-10

10-9

10-8

10-7

0

0.2

0.4

0.6

0.8

1x 10

-10

Increasing KD1

Increasing KC1

10-13

10-12

10-11

10-10

10-9

10-8

10-7

0

1

2

3

4

5

6x 10

-11

10-13

10-12

10-11

10-10

10-9

10-8

10-7

0

0.2

0.4

0.6

0.8

1

1.2x 10

-10

10-13

10-12

10-11

10-10

10-9

10-8

10-7

0

0.2

0.4

0.6

0.8

1x 10

-9

Effects of Varying Parameters on CHIL-6

Increasing KC2


HIL-6 Concentration (M)

Sig

nalin

g C

om

ple

x N

um

ber

(M)

Increasing KD2

10-13

10-12

10-11

10-10

10-9

10-8

10-7

0

0.5

1

1.5

2

2.5x 10

-10

10-13

10-12

10-11

10-10

10-9

10-8

10-7

0

0.2

0.4

0.6

0.8

1x 10

-9

Effects of Varying Receptor Numbers

Increasing LIF receptor numbers


LIF Concentration (M)

HIL-6 Concentration (M)

Sig

nalin

g C

om

ple

x N

um

ber

(M)

Experimental Outline

Getting the parameters for the computational modelCulture of “HSC-enriched” populations under different cytokine concentrationsAssay to quantify HSC self-renewal during culture

Cell Lines

“HSC-enriched” population, using c-kit+Sca-1+lin- cellsIsolation from bone marrow Immunomagnetically removed lin- BM

cells Fluorescent labelling with antibodies Fluorescent-activated cell sorter

(FACS)

Getting the Unknown Parameters…

LIFR and gp130 receptor numbersDissociation and cross-linking rate constants for LIF and HIL-6

gp1

30

gp1

30

KC2KD2

gp1

30

gp1

30

HIL-6

HIL-6

HIL-6

LIF

LIF LIF

LIFR

LIFR

LIFR

gp1

30

gp1

30

KC1

KD1


Parameters: protocol (1)

Radioactive labeling of HIL-6 and LIFSteady state at 37°C Free Cell-bound Internalized

For varying concentrations of ligand

Hilton and Nicola (1992)

Parameters: protocol (2)

Isolated membranes

Detergent-solubilized membranes

KC2KD2

LIFLIF LIF

KC1KD1

LIFKD1 KD2

LIF

Culture Conditions

In suspension: serum-free mediumEach is colony derived from a single cell100 ng/mL FL + 50 ng/mL SCFVarying concentrations of LIF or HIL-6

Adapted from Audet et al. (2001)

Assessment of HSC

Measurement of human engraftment by PCRQuantification of human cells by flow cytometry (6 weeks)Secondary transplantation assay

Culture of human HSC

BM injection

NOD/SCID

Yahata et al. (2002)

Conclusions

gp130 activation plays key role in HSC self-renewal, whether induced by LIF or by HIL-6ESC model is adaptable for HSC systemUnknown parameters can be determined from binding experimentsSwitch-like response of LIF-induced activation is more convenient for clinical applications Renewable source of cells in tissue-replacement

therapies

Selected References

1. Audet, J., Miller, C.L., Rose-John, S., Piret, J.M. & Eaves, C.J. (2001) PNAS 98, 1757-1762.

2. Fischer, M., Goldschmitt, J., Peschel, C., Brakenhoff, J.P.G., Kallen, K.J. Wollmer, A., Grotzinger, J. & Rose-John, S. (1997) Nat. Biotechnol. 15, 142-145.

3. Viswanathan, S., Benatar, T., Rose-John, S., Lauffenburger, D.A. & Zandstra, P.W. (2002) Stem Cells 20, 119-138.

4. Hilton, D.J. & Nicola, N.A. (1992) J. Biol. Chem. 267, 10238-10247.

5. de Wynter, E., et al. (2001) J. Biol. Regul. Homeost. Agents 15, 23-27.

6. Peters, M., Muller, A.M., and Rose-John, S. (1998) Blood. 92, 10:3495-3504.

7. Yahata et al. (2002) Blood.

role of gp130 activation for hsc self-renewal

Documents