Receptors of insulin and IGF complementary
mediate signals for the mammalian cartilage
organogenesis

研究例 5
軟骨形態形成の制御

11○Mariko Ojima and Yohei Hirai
Dept. Bioscience., Grad. Sch. Sci. Technol., Kwansei Gakuin Univ.
Abstract

The effect of insulin on the cell proliferationand the cell motility

ATDC-5 cells treated with insulin
cell aggregation

Alcian blue staining
resting cartilage
cell condensation

IGF-1 insulin

Motility

Proliferationdifferentiated

*

*

*

hypertrophic

0.25
Phase contrast

Alcian blue

2.5day03712 17AbsorbanceThe expression ofa chondrogenic marker (W. B.)
20Area (mm2)0.2What is the role of IGF-1 / insulin signaling on chondrogenesis?

0.15
0.1
0.05

Collagen type ll

1

E13.5 tailbud

controlinsulin

FCS

Insulin activated the proliferation and motility, which wasconsidered to be important for morphogenesis.
3

The effect of exogenous IGF-I / insulin on cartilage morphogenesis in 3D-tailbudwith dysfunctional IGF-IR
10% FCS

0% FCS+insulin+IGF-1

Inhibit IGF-1R

culture medium

membrane

Expand onto culture dish4

Inactivation of both IR / IGF-1R
Dose dependency

Time course

p-Akt

AG538 0 15 30 60 (min)
p-ERK

*1.2

Total ERK

Total Akt

Response to IGF-1 / insulin

p-ERK- IGF-1 insulin
p-ERK

Total ERK

Total ERK

IR

10.8
0.6

10% FCS

0% FCS

Inhibit IGF-1R

0.4

AA

A

B

0.2

Both IGF-1 and insulin react totheir receptor to activate
downstream signalings.
6

The effect of IR knockdown

3Dir
IR+

igf-1rβ-actin

gapdh

W.B.

MOCK

p-Akt

0.80% FCS

Inhibit
IGF-1R

A/B>1

A/B≒1

A/B<10% FCS0% FCS+IGF-1

0% FCS+insulin

5Conclusion
ΔIR / MOCK (%)

RT-PCR

0.9

Inhibition of IGF-1R severely depressed the cartilage formation, which was restored
either by IGF-1 or insulin.

7W.B.

B

10.7

10% FCS

When both IGF-1R and IR were inactivated:
IGF-1=insulin=control (No signal)

Effect of inactivation of IGF-1R

B

0downstream activation (phospholiration of Akt / ERK)

ERK

*A/B (relative)

AG538 0 0.2 0.4 0.8 (μM)

A/B (relative)

Interaction of IR / IGF-1R
IGF-1
Insulin150100
MOCK50ΔIR

Ordinary

Inhibit IGF-1RInhibit IR

IGF-1 Insulin

IGF-1 Insulin

IGF-1 Insulin

0ir

ΔIRigf-1r

IGF-1 Insulin

W.B.

p-Erk

p-IGF-1Rβ-actin

p-IR
p-Akt

2DW.B.

0FCS

3D-culture

Removal ofepidermis

IGF-1R inhibitor

insulin

22D-culture

IGF-1R inhibitor

10Insulin induced chondrogenic
differentiation via condensation.

Trypsinization-1.5

β-actin

The culture of E13.5 tailbuds

Akt

20.5control Insulin induced the differentiation and activated proliferation / motility in chondroprogenitor ATDC-5 cells.
 Attempts were made to clarify the role of IGF-1 / insulin signaling on the cartilage morphogenesis.
When IGF-1 receptor (IGF-1R) was inactivated,• the normal cartilage morphogenesis was prevented in 3D-tailbud, which was partly restored
by IGF-1 or insulin.
• insulin activated downstream more than IGF-1 does.

IGF-1R*3

IGF-1R

p-ERK-IGF-1R+

When IR was knock-downed : IGF-1 = insulin >> control (No signal)p-Erk

9β-actin

The regulation of the expression of IRby IGF-1 / insulin
Supplementary…

 Ordinary : IGF-1 > insulin >> control (No signal)
 When IGF-1R was inactivated :insulin > IGF-1 >> control (No signal)
8W.B.
IRβ-actin

2
Relativev.s. β-actin

downstream activation(phospholiration of Akt / ERK)

IGF-1RIR

IGF-1RIR

IR

When IR was knock-downed : IGF-1R expression was up-regulated
downstream activation (phospholiration of Akt / ERK)

p-AktIR

1.5
10.50-

IGF-1R / IR signaling plays a key role not only on
growth / differentiation but also on themorphogenesis of mammalian cartilage.
IGF-1 and insulin can bind to both of their
receptors to activate the downstream signaling.
IGF-1R and IR transduce signals in acomplementary manner.

IGF-1 insulin

Insulin suppressed, while IGF-1 increased, the expression of IR.

1011