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

研究例 3毛の性状制御○Shunsuke Fujimoto , Yohei Hirai
Dep. Bioscience., Grad. Sch. Sci. Technol., Kwansei Gakuin Univ.

1.Introduction

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

2. The localization of Hacl-1 proteinin the vibrissals hair follicles

The major components of the hair are keratins and keratin
associate-proteins (KAPs). KAPs have been supposed to play aroles in the keratin filamentous assembly so as to maintain thephysical 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, wetested the effect of Hacl-1 overexpression in cultured hair follicle.
We also investigated the molecular characteristics andsubcellular localization of Hacl-1 and its binding partner.

Immunostaining

Immunostaining
Hacl-1

Nude(𝐹𝑜𝑥𝑛1𝑛𝑢 )W.T(C3H)

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

Journal of Investigative Dermatology 102, 716-720 1994

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

Electroporation and organ culture
CMVpromotor(Day5)

gfp

internalcontrol

Hacl-1GFP

Transgene

IRES

Upper lip skin(13g.d.)normalW.B.

Day5

abnormalanti-T7
Hacl-1GFP

β - actin

Hacl-1

Hacl-1 probe

Hair keratin

normalCysts-like structures with keratinized debris

50%0%Hacl-1GFP

Hacl-1 overexpression

HA-taghacl-1

Immunostaining

Negative control

streptavidin

W.B.

Binding analysis with Hacl-1 probeAbsorbance* Hacl-1 probe* : P < 0.005

Hacl-1 probe

Negative control(GFP probe)Hacl-1 probe-2Me

Hacl-10.06

170.04

streptavidin

Nude(𝐹𝑜𝑥𝑛1𝑛𝑢 )W.T(C3H)

W.T(C3H)

Hacl-1 formed dimer via S - S bonds.

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

CBB: type II keratin: type I keratin

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

HA(Hacl-1)

hacl-1

W.B.
Proteasome inhibitor
anti-HA-+Hacl-1

Keratinocyte(HaCaT)

YZ

Localization of Hacl-1 in the keratinized
HaCaT cells.

The exogenous Hacl-1 was localized
in the cell nuclei as granules.
100%
80%

n=560%

lipofection

0%+-The NES tag exported the Hacl-1 granules to cytosol.
NES : nuclear export signal－(CDLTLRELPPLQL)

transient

HaCaT cells stably
introducedwith Hacl-1 geneHacl-1 was actively degraded by theproteasome protein-eliminating system.

Irregular
expression

normal→The expression pattern of the Hacl-1 may be controlled
by keratinization progression.
・The binding partner of Hacl-1 localized in hair cortex andthe 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.

HA(Hacl-1)

Hacl-1

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

stablecytosol

20%
NES-tag

Calcium Ionophore
A23187

Internalcontrol# : non-specific band

・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.
Hacl-1overexpression

nucleus40%

Keratinocyte(HaCaT)Internal
control

GFP

9. Conclusion

lipofection

XZ

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

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

Immunostaining

17

Internalcontrol

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

HA(Hacl-1)

38

Hacl-10XY

West-Western analysis
with Hacl-1 probe

+38

0.08

IRES

anti-HAGFP probe

0.02

lipofection

T7
hacl-1

gfp

0.1

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

Hairless

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

0.12

Negative control(GFP probe)

Nude

7. The cohesive self-assembly of Hacl-1

streptavidin

n=6

HA-taghacl-1
NES

W.T

100%

Hacl-1

Histochemistry with Hacl-1 probe

streptavidin

West-Western analysis with Hacl-1 probe

Keratinocyte(HaCaT)

0%

abnormalstreptavidin

HA-taghacl-120%

Bindingpartnerbiotin

Hacl-1 probe40%

Hacl-1 overexpression caused abnormal follicular structures.

Preparation of Hacl-1 probebiotinylation
Blocking active SH

60%

n = 19

6. Detection of the binding partner of Hacl-1

Biotinylated
Hacl-180%

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

Hacl-1100%

Abnormal hair follicles
with Hacl-1 overexpression

abnormalPhenotypic appearance (%)

Hacl-1Day1

n = 12The concentrically expression pattern
was perturbed in nude mice.

T7
hacl-1

Day3

normal

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

W.B.

Upper lip skin(13g.d.)abnormalHairless(HR-1)

Hacl-1 localized specifically in the hair cortex.

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

The rate of hair follicles (%)

AHF
Hacl-1empty
SHacl-1 expression was regulated by keratinization.

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

SAssociation without disulfide bonds

S-S bridges

Hacl-1
type I keratin
type II keratin