An important role and the strict expression control of Hacl-1, a hair follicle specific KAP

○Shunsuke Fujimoto , Yohei Hirai Dep. Bioscience., Grad. Sch. Sci. Technol., Kwansei Gakuin Univ.

The major components of the hair are keratins and keratin associate-proteins (KAPs). KAPs have been supposed to play a roles in the keratin filamentous assembly so as to maintain the physical strength of hair shaft. Previously, Hacl-1, a member of KAPs family, was detected exclusively in the anagenic hair follicle, however, little is known about this protein. In this study, we tested the effect of Hacl-1 overexpression in cultured hair follicle. We also investigated the molecular characteristics and subcellular localization of Hacl-1 and its binding partner.

1.Introduction 2. The localization of Hacl-1 protein in the vibrissals hair follicles

Journal of Investigative Dermatology 102, 716-720 1994

The previous study demonstrated that Hacl-1 mRNA is expressed in the anagenic hair.

Immunostaining

Hacl-1 localized specifically in the hair cortex.

3. The expression pattern of Hacl-1 in mutant mice

Immunostaining

W.T(C3H) Nude(𝐹𝑜𝑥𝑛1𝑛𝑢)

Hairless(HR-1)

The concentrically expression pattern was perturbed in nude mice.

0%

20%

40%

60%

80%

100%

W.T Nude Hairless

abnormal normal

4. The expression of Hacl-1 upon the hair follicle formation in culture

gfp

hacl-1

T7

IRES

CMVpromotor

Hacl-1

β - actin

anti-T7

0%

50%

100%

Hacl-1 GFP

abnormal normal

n = 19

GFP Hacl-1

Organ culture

W.B.

Transgene GFP Hacl-1

Hacl-1 overexpression caused abnormal follicular structures.

Upper lip skin (13g.d.)

(Day5)

normal

abnormal

6. Detection of the binding partner of Hacl-1

8. The subcellular localization of exogenous Hacl-1 in non-trichocyte cells

Upper lip skin (13g.d.)

Day1 Day3 Day5

Hacl-1

internal control

W.B.

Expression of Hacl-1 was accompanied by the hair follicle formation.

5. The Effect of Hacl-1 on the hair follicle formation

Electroporation and organ culture Abnormal hair follicles with Hacl-1 overexpression

Hair keratin

Cysts-like structures with keratinized debris

Hacl-1 overexpression

abnormal

normal

n = 12

Hacl-1

Hacl-1

biotin

Blocking active SH biotinylation

Histochemistry with Hacl-1 probe

streptavidin streptavidin

West-Western analysis with Hacl-1 probe

Hacl-1 interacted with type I hair keratin in hair cortex without disulfide bridges.

Negative control

Hacl-1 probe

Preparation of Hacl-1 probe

Hacl-1 may have the ability to form dimeric products with disulfide bonds.

transient stable

Proteasome inhibitor -

XY XZ

YZ

HA(Hacl-1)

Keratinocyte (HaCaT)

9. Conclusion

streptavidin

streptavidin

W.T(C3H) Nude(𝐹𝑜𝑥𝑛1𝑛𝑢) W.T(C3H)

Hacl-1 probe

Hacl-1 probe

Immunostaining

7. The cohesive self-assembly of Hacl-1

Hacl-1

2Me - +

17

38

17

38

Internal control

Hacl-1 formed dimer via S - S bonds.

W.B.

gfp

hacl-1

T7

IRES

hacl-1

HA-tag

West-Western analysis

with Hacl-1 probe

Hacl-1 GFP

Hacl-1 probe couldn’t bind to Hacl-1 protein without active cysteines.

Internal control

Internal control

Immunostaining

hacl-1 HA-tag

W.B.

HA(Hacl-1) hacl-1

HA-tag

NES

NES : nuclear export signal

The NES tag exported the Hacl-1 granules to cytosol.

－(CDLTLRELPPLQL)

Calcium Ionophore A23187 HA(Hacl-1) hacl-1

HA-tag

Hacl-1 expression was regulated by keratinization.

Association without disulfide bonds

S S Hacl-1

type I keratin type II keratin

S-S bridges

The binding partner of Hacl-1 in Nude mice appeared to distribute similarly to WT mice.

The exogenous Hacl-1 was localized in the cell nuclei as granules.

Localization of Hacl-1 in the keratinized HaCaT cells.

Hacl-1 was actively degraded by the proteasome protein-eliminating system.

n = 5

Expression and localization of Hacl-1 was regulated by keratinization.

anti-HA

Hacl-1

Internal control

anti-HA

0

0.02

0.04

0.06

0.08

0.1

0.12

Binding analysis with Hacl-1 probe

The binding partner specifically localized in the hair and was probably keratin type I. Hacl-1 may interact with other KAPs.

*

n = 6 * : P < 0.005

HA(Hacl-1) ・The binding partner of Hacl-1 localized in hair cortex and the intermolecular interaction didn’t require active cysteine. (It may be hair keratin type I) ・Hacl-1 may have the ability for the cohesive self-assembly. → Hacl-1 may confer the mechanical strength upon hair keratin IFs.

Hacl-1

AHF Hacl-1

The

rate

of

hai

r fo

llicl

es (

%)

Ph

en

oty

pic

ap

pea

ran

ce (

%)

Ab

sorb

ance

lipofection

lipofection

Hacl-1 protein was expressed in the lower and middle portion of the cortex.

HA(Hacl-1) T7(Hacl-1)

Binding partner

Biotinylated Hacl-1

streptavidin

Hacl-1 probe Negative control (GFP probe)

CBB

Negative control (GFP probe)

: type II keratin : type I keratin

Hacl-1 probe GFP probe

Calcium Ionophore A23187

HaCaT cells stably introduced with Hacl-1 gene

Keratinocyte (HaCaT)

Keratinocyte (HaCaT)

lipofection

0%

20%

40%

60%

80%

100%

+ -

nucleus

cytosol

NES-tag

・The expression pattern of Hacl-1 was perturbed in nude mice. ・Hacl-1 overexpression caused abnormal morphogenesis/differentiation of the hair follicle. →Expression control of Hacl-1 protein may be crucial for normal development of hairs and their follicles.

→The expression pattern of the Hacl-1 may be controlled by keratinization progression.

Hacl-1 overexpression normal

Irregular expression

Hacl-1 empty

#

# : non-specific band

+

研究例 3

毛の性状制御