Go J. Yoshida M.D.,Ph.D. (E-mail:goscm@tmd.ac.jp)Dept. of Stem Cell Biology, Medical Research Institute

Tokyo Medical and Dental University, Tokyo, Japan

Anatomy and Histology of Lungs

Basal stem cells are committed to luminal cells.

K5, K14, ITGA6, NGFR,p63

Identification of ABSC markers by 3D culture

K5, K14, ITGA6, NGFR,p63

Acetylated alpha tubulin is present in various microtubule structures and plays a role in

stabilizing the structures of all microtubules

cilia

PNAS (2009)

The protective role of Nrf2 for the lungs

ROS is a double edged sword!!!

Low-moderate ROS level

Signal transduction

Proliferation

Metabolism

Moderate-high ROS level

Cell death

Senescence

Differentiation

Notch signal depends on the cell-to-cell interaction.

Working Hypothesis

Airway basal stem cells (ABSCs) harbor the mechanism to

respond to redox stress in the micro-environment,

otherwise they would not show the self-renewal phenotype.

COPD

(chronic obstructive

pulmonary disease) Cancer

Heterogeneous ROS level of airway basal stem cell

(ABSC) reflects the sphere formation potential.

CC10: differentiation marker

ROS is a double-edged sword for the self-renewal

and proliferation potential of ROS(low)-ABSC.

ROS flux: shift from ROS(low) to ROS(mediate)

Alteration of ROS changes the expression of K5

(ABSC marker) and K8 (early progenitor cell marker).

Chemical compound for injury of airway epithelium.

Polidocanol

used as a sclerosant, an irritant injected to treat varicose veins, under the trade names Asclera

Normal Repair, otherwise

Fibrosis

Hyperplasia

� γ-tubulin : the marker of the mitotic spindle angle

� atypical protein kinase C (aPKC): the polarity marker

Most of the epithelial cells orient the spindle along the plane of the epithelium (planar

orientation) for mitosis to produce two identical daughter cells (symmetric division).

The correct orientation of the spindle relies on the interaction between cortical

polarity components and astral microtubules

symmetric or asymmetric cell fates

Spindle orientation influences on the generation of

symmetric or asymmetric cell fates

ROS(high)-ABSCs undergo asymmetric cell division

48hr after injury.

ROS(low)-ABSCs mainly undergo symmetric cell

show asymmetric cell division 48hr after injury.

ROS(low)-ABSCs mainly undergo symmetric cell

division 24hr after injury, while ROS(high)-ABSCs also

show asymmetric cell division 48hr after injury.

symmetric

symmetric

asymmetric

ROS(high) reduces

cell division.

ABSC-derived spheroids show asymmetric or

symmetric cell division depending on ROS level.

The rate of ABSC proliferation is consistent with

increased ROS level during Day3-7.

ABSC show asymmetric cell division with high ROS

level 48hr after injury to express K8 instead of K5.

Not only ABSCs but also lung cancer cells show

significant increase of transition from G1 to S phase.

A549

(lung adenocarcnioma cell line)

Increased expression of NQO-1, one of the target

flux and spheroid proliferation.

Increased expression of NQO-1, one of the target

molecules for Nrf2, is positively correlated with ROS

flux and spheroid proliferation.

The expression level of intra-nuclear Nrf2 and NQO1

was higher during the repair of tracheal epithelium.

ABSC; purple

Early progenitor; green

Nrf2-dependent anti-oxidant machinery is critical to

maintain the homeostasis of the tracheal epithelium.

Notch+/K5+/BrdU+: green

Notch+/K5+/BrdU-: orange

Notch-/K5+/BrdU+: yellow

Notch pathway is maintained by Nrf2 and important

for the self-renewal of ABSC.

Notch effector proteins are up-regulated during the

repair process.

K5-CrePR1::Rosa NICD

K5-NICD

WT

K5-NICD

+ injury

Chemical injury in K5-NICD mice leads to excessive

self-renewal of ABSC, resulting in the hyperplasia.

DBZ (Notch

inhibitor)

WT + injury

Nrf2 depletion fails to induce the expression

of Notch signaling effectors such as Hey1.

Notch reporter assay shows the association between

Nrf2 and Notch1.

SFN-responsive

Nrf2 activation

constitutive active Nrf2

Nrf2 directly binds to ARE consensus

sequences within Notch1 promoter.

Nrf2 directly binds to ARE consensus

sequences within Notch1 promoter.

EMSA (Electrophoresis Mobility Shift Assay)

Gain-of-functional mutation of Nrf2

significantly increases Notch1 expression .

Conclusion

ROS-mediated regulation of ABSC

ROS-Nrf2-Notch1 axis is important for lung stem cells to

respond to changes in surrounding ROS levels.