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Keratin is not only a Structural Protein in Hair:
Keratin-mediated Hair Growth
Seong Yeong An
Kyung Hee University
Eun Ji Choi
Konkuk University
So Yeon Kim
Kyung Hee University
Se Young Van
Kyung Hee University
Han Jun Kim
Konkuk University
Jae-Hyung Lee
Kyung Hee University https://orcid.org/0000-0002-5085-6988
Song Wook Han
KeraMedix Inc
Il Keun Kwon
Kyung Hee University
Chul-Kyu Lee
Chemon Inc.
Sun Hee Do
Konkuk University
Yu-Shik Hwang ( yshwang@khu.ac.kr )
Kyung Hee University
Article
Keywords: Keratin , hair, intradermal injection, outer root sheath cells,
DOI: https://doi.org/10.21203/rs.3.rs-101358/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Keratin is known to be a major protein in hair, but the biological function of keratin in hair growth is
unknown, which led us to conduct a pilot study to elucidate biological function of keratin in hair growth
via cellular interactions with hair forming cells. Here, we show hair growth is stimulated by intradermal
injection of keratin into mice, and show that outer root sheath cells undergo transforming growth factor-
β2-induced apoptosis, resulting in keratin exposure. Keratin exposure appears to be critical for dermal
papilla cell condensation and hair germ formation as immunodepletion and silencing keratin prevent
dermal papilla cell condensation and hair germ formation. Furthermore, silencing keratin in mice resulted
in a marked suppression of anagen follicle formation and hair growth. Our study imply a new nding of
how to initiate hair regeneration and suggests the potent application of keratin biomaterial for the
treatment of hair loss.
Introduction
Keratin is a cytoskeletal protein that forms intermediate laments within epithelial cells and participates
in maintaining the strength of the cells1, and keratins are also the major proteins deposited inside the hair,
contributing to its mechanical strength2. Human hair consists of three main layers: the medulla in the
center of the hair, the cortex surrounding the medulla, which contains ber mass mainly consisting of
keratin protein, and the cuticle, the outer layer of the hair shaft3. During hair growth, dermal papilla (DP)
cells secrete various paracrine factors to induce cell migration of stem cells from the bulge region of the
outer root sheath (ORS) to the upper region of the follicle, and the migrated cells become transit
amplifying cells, which then undergo differentiation into the matrix cells. Growth of the hair is initiated by
cortical cells differentiated from matrix cells located in the follicle bulb region, and a large amount of
keratin is synthesized mainly in the cortex4–6. Deposition and rearrangement of keratin lament is
followed by the assembly of keratin-associated proteins and intracellular deposited keratin in spindle-
shaped epithelial cells of the cortex, and the assembly is stabilized by the formation of inter- and intra-
molecular disulde bonds7. At the stages of the anagen-catagen transition of the hair cycle, apoptosis of
cells appears in the epithelial strand, and then the apoptotic cells are phagocytosed by macrophages and
neighboring epithelial cells, but simultaneously cellular organelles are degraded and removed. Ultimately,
keratins remain the main proteins in the hair8–11. Such a massively deposited keratin in hair has recently
been considered one of the potent biomaterials due to its good biocompatibility based on human origin
and an abundant source of 300,000 tons of annually wasted hair worldwide. Keratin has been widely
used for the development of various biomaterials for biomedical applications in the eld of wound
healing, nerve regeneration and bone regeneration3.
In our previous study, mice models with full-thickness dorsal excisional wounds were used to assess the
effect of keratin-based hydrogel on wound healing12. Interestingly, hair growth was found only in areas
treated with keratin hydrogel with accelerated wound healing, which led us to study the biological
function of hair-derived keratin in hair regeneration. In this study, keratin was extracted from human hair,
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and mice model show hair follicle formation and following hair growth was promoted by intradermal
injection of hair-derived keratin, and then the underlying biology of keratin to stimulate was studied by
studying the interaction of keratin with DP cells and ORS cells, which are known as the main types of
cells that regulate hair growth and regeneration. Hair growth was found to be modulated by keratin-
mediated DP condensation and hair germ (HG) formation of ORS cells. Such DP condensation and HG
formation was mediated by spatial release and deposition of keratin from the apoptotic ORS cells
following TGFβ2-induced ORS cell apoptosis and caspase-mediated degradation of keratin. Our pilot
study represents that keratin is not only a structural protein of hair but also a factor to induce hair
regeneration.
Results
Intradermal Injection of Hair-derived Keratin Promotes Hair
Growth
First, we performed
in vivo
experiments on mice to evaluate keratin-mediated hair growth by injecting
hair-derived keratin into hair-removed dorsal skin area. The hairs on dorsal skin of C57BL/6 mice were
removed by hair depilation cream with clipping for hair follicle synchronization, and then keratin was
injected. After 14 days of keratin injection, we found that the hair growth was promoted (Fig.1A) with
higher formation of anagen follicles than in non-treated mice (Fig.1B-D), and the sizes of hair follicles
were found to be increased upon keratin injection (Fig.1E, F). Only single injection of keratin resulted in
almost equivalent or slightly higher hair growth in mice compared to minoxidil, applied every day for 14
days. Such keratin-mediated hair growth was also conrmed in a separate mouse study, and hair growth
and hair follicle formation in keratin-injected C57BL/6 mice with different concentrations of 0.5 and 1.0
(w/v) % of keratin were analyzed for the number and stage of hair follicles. The promoting effect of
keratin injection on hair growth was veried, and there were no signicant differences in the number and
stage of the formed hair follicles in mice injected with different concentrations of keratin (Supplementary
Fig.1A, B).
Keratin Induces Condensation of DP Cells and Germ
Formation of ORS Cells In Vitro
To understand how injected keratin induces hair follicle formation and hair growth, we studied the
extracellular interaction of keratin with DP and ORS cells, which are known to be main cells participating
in hair follicle formation4–6, 13. The most distinct characteristic of DP cells exposed to keratin for 3 days
was condensation to form spherical aggregates (Fig.1G, H) with high expression levels of β-catenin,
SOX2, CD133, and alkaline phosphatase (ALPase) (Fig.1H), which is a molecular identity signature
reecting hair inductive property of DP cells14,15, and high expression levels of FGF7, FGF10, and BMP6
(Fig.1H), reecting paracrine factors for controlling hair growth16 (keratin-mediated condensation of DP
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cells on day 1 and time-lapse images are presented in Supplementary Fig.2A, C). However, the growth of
DP cells was suppressed upon exposure to keratin (Supplementary Fig.2B). Such keratin-mediated
condensation of DP cells was observed, when cells were seeded at different cell densities, and on
matrigel (Supplementary Fig.3, 4), and no signicant difference in DP cell condensation were not found
when DP cells were treated with different keratin concentrations (Supplementary Fig.5). RNA sequencing
analysis showed a downregulation in expression levels of genes associated with cell division and an
upregulation of mRNA-encoding proteins related to integrins, growth factors, migration, and extracellular
matrix organization (Supplementary Fig.6A, B). In addition, the effect of keratin on maintaining the DP
cell condensation was analyzed. The DP cell spheroids showed higher levels of gene expression,
indicating hair inductive property of DP cells as compared to a DP cell monolayer (Fig.2A), and the
spherical shape of DP cell aggregates was constantly maintained, showing high levels of molecular
expression of β-catenin, SOX2, CD133, ALPase, FGF7, FGF10, and BMP6 (Fig.2B-D) in the presence of
keratin. ORS cells showed the formation of colony within a few hours of exposure to keratin and began to
temporarily proliferate as early as on day 1 of cultivation, subsequently forming strand-like extended
structures by day 3 (Fig.3A, B). High β-catenin expression, known to occur during migration and further
differentiation of stem cells in the ORS region6, and a local cell population expressing P-cadherin, known
to be a marker of secondary HG formation17,18, were observed along with extended structures in keratin-
treated ORS cells (Fig.3C, low-magnication images of β-catenin and P-cadherin expression in keratin-
treated ORS cells are presented in Supplementary Fig.7). In addition, keratin-treated ORS cells showed
lower expression levels of CD34 compared to untreated ORS cells, but maintained high levels of SOX9
expression (Fig.3C). Furthermore, RNA sequencing analysis of keratin-treated ORS cells revealed
upregulated mRNA expression levels of acidic hair keratins, mainly KRT31, KRT33B, KRT34, and KRT37
(Supplementary Fig.8A). We also observed an increased molecular expression of KRT34 and β-catenin
(Supplementary Fig.8B). These ndings imply hair keratin-mediated alterations in molecular and gene
expression proles indicating germ formation and further differentiation of ORS cells.
TGFβ2 induces Apoptosis of ORS cells and generates
Keratin Release and Deposition, mediating Condensation of
DP Cells
The ndings described above demonstrated that extracellular interaction of keratin induced the
condensation of DP cells and the formation of P-cadherin expressing germs of ORS cells, which led us to
ask the question whether the observed pattern of the interaction of DP and ORS cells with keratin might
be related to a biological event that occurs during hair cycling. During the anagen-catagen transition
stage, TGFβ2 is synthesized from DP cells stimulated by dihydrotestosterone, and is spatiotemporally
localized in the lower part of the hair bulb at the catagen stage, thus suppressing the proliferation of
epithelial cells, but inducing caspase-mediated apoptosis11. Therefore, we hypothesized that exposure of
keratins from apoptotic ORS cells during hair cycling might drive DP cell condensation and secondary HG
formation through extracellular interaction with DP and ORS cells. To address this question, we induced
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apoptosis of ORS cells by treating with TGFβ2 and characterized microenvironmental changes, such as
the release or deposition of keratin. In our study, apoptosis array analysis showed upregulation of
apoptosis-related markers, including Bax, caspase-3, cytochrome C, and SMAC in ORS cells treated with
TGFβ2 (Fig.4A, Supplementary Fig.9). Extended structures composed of spindle-shaped ORS cells
developed only in the presence of TGFβ2; annexin V and tunnel positive apoptotic cells were mainly
found in the extended structures of TGFβ2-treated ORS cells. High expression levels of caspase-3 and
massive deposition of keratin were observed along the extended structures (Fig.4B). To determine
whether the released or deposited keratin from TGFβ2-induced apoptotic ORS cells could inuence DP
cell condensation, the condensation activity of DP cells was tested by direct contact co-culture and
culture in conditioned media. Local condensation of DP cells with the formation of spherical cell colonies
was observed in the concentric region of the extended structure in TGFβ2-treated ORS cell layers (Fig.4C,
Supplementary Fig.10A). The conditioned medium collected from TGFβ2-treated ORS cell cultures
contained relatively higher levels of keratin, and the DP cell condensation to form spherical cell
aggregates were distinctly improved in the DP cell culture under the conditioned media (Fig.4D,
Supplementary Fig.10B). These results indicate that the deposition or release of keratin from TGFβ2-
induced apoptotic ORS cells could be a regulator of the induction of DP cell condensation.
Keratin Release and Deposition through Caspase-6-
mediated Keratin Degradation stimulates Condensation of
DP Cells
Keratin fragmentation occurs during apoptosis of epithelial cells19; intracellular insoluble keratin is
disposed during apoptosis via fragmentation into soluble fragments by caspases20. In our study,
apoptosis array analysis showed a two-fold increase in caspase-3 expression levels in TGFβ2-treated
ORS cells, and it was reported that type I keratin, including hair keratin, contains a cleavage site, VEVD, for
caspase-621. When hair keratin was digested with caspase-3 and caspase-6, fragmented keratin was
generated only in hair keratin digested by caspase-6 (Fig.5A), and higher gene expression and protein
expression levels of caspase-6 and its cleaved form (active caspase-6) were observed in TGFβ2-treated
ORS cells (Fig.5B, C). This nding implies that the release and deposition of keratin from TGFβ2-treated
ORS cells through caspase-6-mediated degradation can inuence the DP cell condensation. Hence, to test
this, we silenced the expression of caspase-6 gene in ORS cells and examined the levels of released
keratin (Fig.5D). We observed lower levels of released keratin in caspase-6-silenced ORS cells in the
presence of TGFβ2 (Fig.5E) and lower condensation activity of DP cells in conditioned media collected
from caspase-6-silenced ORS cell culture and in co-culture on the caspase-6-silenced ORS cell layer in the
presence of TGFβ2 (Fig.5F-H). Caspase-6-silenced ORS cells developed relatively spread out structures
even in the presence of TGFβ2, which were different from the extended structures of TGFβ2-treated non-
silenced ORS cells. Tunnel positive apoptotic cells were found in both the spread and extended structures,
and it was found that keratin deposition is inuenced by caspase-6 expression levels (Fig.5H,
Supplementary Fig.11). We then performed an immunodepletion assay to obtain direct evidence for
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keratin-mediated condensation of DP cells by removing keratin from the conditioned medium of TGFβ2-
treated ORS cells using a column containing anti-human type I + II hair keratin antibody-conjugated
beads. The elimination of keratins in conditioned media was conrmed (Fig.6A), and there was no
substantial difference in the growth factors contained in the conditioned media before and after
immunodepletion (Fig.6B, Supplementary Fig.12). The immunodepletion assay showed that the removal
of keratins resulted in suppressed DP cell condensation (Fig.6C), and collectively these data reected the
functional role of keratins in DP condensation.
Spatial Keratin Deposition by TGFβ2-induced Apoptotic ORS
cells induces Germ Formation
Along with keratin-mediated condensation of DP cells, to understand how the release or deposition of
keratin during TGFβ2-induced ORS cell apoptosis triggers germ formation, the keratin released from
TGFβ2-induced ORS cells were tested for its ability to induce germ formation. Contrary to DP cell
condensation, the released keratin in conditioned media was not effective in generating P-cadherin
expressing cell population, which was proved by immunodepletion assay (Supplementary Fig.13). This
result prompted us to ask whether secondary HG formation in cells expressing P-cadherin is inuenced by
the spatially deposited keratin caused by spatiotemporal apoptosis of ORS cells. TGFβ2 expression,
which was restricted to the outermost ORS cell layer in the anagen phase, was reported to be upregulated
spatiotemporally in the boundary region between germinal matrix cell and the DP cell in the lower bulb
region during the anagen-catagen transition8. Therefore, to study the spatial deposition of keratin from
TGFβ2-induced apoptotic ORS cells and its effect on the germ formation of ORS cells, the time-course
effect of TGFβ2 treatment on protein expression levels of caspase-6, keratin deposition, and germ
formation were characterized. The extended structure progressively developed in the TGFβ2-treated ORS
cell layers over the cultivation time, and the P-cadherin expressing germ was spatially developed in the
TGFβ2-treated ORS cell layers (Supplementary Fig.14). Immunocytochemical staining of the TGFβ2-
treated ORS cell layers showed that a population of RUNX1 and P-cadherin-positive cells, representative
markers of germ formation10,22−24, emerged in the concentric region of the extended structure, and the
caspase-6-expressing apoptotic cell population and keratin-deposited area also increased over time in the
extended structure (Supplementary Fig.14). Next, to consider the effect of spatial deposition of keratin on
the formation of P-cadherin expressing germ
in vitro
, the expression of KRT31/KRT34 in ORS cells was
silenced by siRNA transfections. The downregulated molecular expressions of keratin in both conditioned
media and ORS cells via KRT31/KRT34-silencing were characterized (Fig.6D). Contrary to the well-
developed stranded structure in the ORS cell layers, the KRT31/KRT34-silenced ORS cells did not form an
extended structure even in the presence of TGFβ2, and keratin deposition and emergence of RUNX1 and
P-cadherin expressing ORS cell population were markedly suppressed in the KRT31/KRT34-silenced ORS
cell culture in the presence of TGFβ2 (Fig.6D).
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In Vivo Silencing of Keratin Expression Suppresses Anagen
Hair Follicle Formation and Hair Growth
Combining with all
in vitro
data from the studies of extracellular interaction of keratin, the release and
deposition of keratin from TGFβ2-mediated apoptotic ORS cells have shown a pivotal role in controlling
DP condensation and HG formation. Finally, to determine whether the silenced KRT31/KRT34 expression
can suppress hair follicle formation and hair growth
in vivo
, the keratin expression in mice was
temporarily silenced by intravascular lipofectamine-mediated delivery of KRT31/KRT34 siRNAs. RT-PCR
analysis showed effective KRT31/KRT34 silencing, as indicated by downregulation of KRT31/KRT34
mRNA expressions on day 7 (Fig.7A), and it was found that the KRT31/KRT34 silencing signicantly
inhibited hair growth activity compared to the control (Fig.7B). Notably, the dysregulation of the hair
follicle cycling was observed in KRT31/KRT34 silenced mice; histological analysis of hair follicle sections
showed a strong suppression of the formation of anagen follicles, with no appearance of anagen follicles
in 56% of skin tissue sections of KRT31/KRT34 silenced mice on day 7 (Fig.7C). An anagen bulb
containing a population of cells expressing P-cadherin was hardly seen in immunohistological sections
of KRT31/KRT34 silenced mice (Fig.7D, Supplementary Fig.15A). In contrast, an additional injection of
hair-derived keratin after KRT31/KRT34 siRNA transfection allowed the hair follicles to enter the anagen
phase and regrow hair, similar to the controls. No obvious histological differences were found in the
formation of hair follicles and hair growth between control skin and keratin-injected skin of KRT31/KRT34
silenced-mice after 2 weeks (Fig.7E). Furthermore, the formation of P-cadherin-positive germs and strong
expression of β-catenin were observed in the region of anagen hair follicles in sections of control skin and
keratin-injected skin of KRT31/KRT34 silenced-mice (Fig.7D, Supplementary Fig.15A); strong staining for
KRT34 was found in the ORS region surrounding the DP, which corresponds to the caspase-6-positive
region (Supplementary Fig.15B). Interestingly, it was found that the region stained positively for caspase-
6, KRT34, and P-cadherin move upward into the hair shaft region of the expanded hair follicles
(Supplementary Fig.15B).
Discussion
Hair loss disorders, such as androgenetic alopecia, have been common these days, leading to the
development of new pharmaceuticals for patients with hair loss. In the present study, keratin, the main
structural protein in hair, is proposed as a new candidate for a therapeutic treatment of hair loss. It is
shown here that an intradermal injection of human hair-derived keratin promotes hair growth with the
enhanced formation of anagen hair follicle and an increase in the size of hair follicles. Hair growth is
controlled by the interactions between two distinct cell types: mesenchyme and epithelial cells, while DP
and stem cells from the bulge region of ORS participate in hair follicle formation4–6, 13. The
in vivo
injected keratin-mediated follicle formation and hair growth could be associated with keratin-mediated
DP cell condensation and germ formation via cellular interaction with keratin, as evidenced by our results
of strong expressions of various signature molecules, such as β-catenin and P-cadherin, highly expressed
in hair follicle formation4–6, 13, in keratin-treated DP cell and ORS cell culture. The secondary HG
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expressing P-cadherin emerges at the end of the catagen and at the beginning of the telogen and leads to
the rst stage of hair regeneration17,18. The interaction of P-cadherin with β-catenin plays an important
role in maintaining the anagen phase of the hair cycle25, and the β-catenin expressing migrated cells from
the bulge region of the ORS undergo temporal proliferation and further differentiation in the follicle
region6. Keratin-treated ORS cells showed morphological change, such as spindle shape with strong
expression of β-catenin and downregulated molecular expression of CD34, and a population of P-
cadherin expression cells emerged. It was reported that CD34-positive stem cells convert directly into P-
cadherin expressing HG cells23. Hair regeneration is processed by hair follicles undergoing repeated
cycles of anagen (hair growth stage), catagen (regression stage), and telogen (rest stage)26. During the
late catagen and early telogen phases, secondary HG progressively appears at the base of the follicular
epithelium; at that point, HG cells form a cell cluster and become activated to begin hair regeneration10.
During these phases, DP cells undergo condensation to form a follicular papilla beneath secondary HG.
The interaction between the DP condensate and secondary HG leads to the formation of new hair follicles
by enveloping the DP with the downwardly extended epithelial cells10,22,27. However, in spite of recent
considerable progress in understanding cellular interactions to control hair growth, it is not clear how
secondary HG formation and the DP condensation, the key biological events causing hair regeneration,
are initiated at the beginning of a new hair cycle.
Considering
in vivo
an
in vitro
results of keratin-mediated hair growth and keratin-mediated cellular
change, keratin might not be only a structural component in hair, but might have biological function in
hair growth, and biological phenomena such as keratin-mediated DP cell condensation and germ
formation might be closely related to a biological process that occurs during hair cycling. Hence, keratin-
mediated condensation of DP cells and the formation of P-cadherin germs, assessed by
in vitro
cell study,
led us to explore the biological function of keratin in hair regeneration as a pilot study. At the anagen-
catagen transition stages of hair cycle, the local deposition of TGFβ2 in the lower region of the follicle is
restricted due to the spatiotemporal secretion of TGFβ2 produced by DP cells11, which is consistent with
the spatial gradient of apoptosis of epithelial cells9. Spatiotemporally localized TGFβ2 induces apoptosis
of ORS cells in the lower part of the hair bulb at the catagen stage, resulting in the expression of caspase
and its-mediated the fragmentation of insoluble keratin into soluble keratin fragments8 − 11,19. Although
the relationship between TGFβ2 expression and the intrinsic property of DP cells related to condensation
is not well known, our data showed that TGFβ2 expression was downregulated during DP cell
condensation and rapidly upregulated during the dispersion of condensed DP cells (Supplementary
Fig.16A, B). Mesenchyme condensation such as dermal condensates is promoted by BMP signaling and
transient downregulation of TGF-β signaling, showing an antagonistic relationship16,28,29. The condensed
DP cells might maintain the property of DP cells27 to drive hair growth by releasing paracrine factors,
which can induce stem cell activation and differentiation during the anagen phase, and may induce
spatiotemporal apoptosis of adjacent ORS cells via increased expression of TGFβ2 during the anagen to
catagen transition. In our study, it was shown that local DP cell condensation and germ formation in
TGFβ2-induced apoptotic ORS cells depend on the exposure of keratin via caspase-6 expression and
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consequently its-mediated keratin release or deposition, as evidenced by the suppressed DP cell
condensation and germ formation in caspase-6-silenced or KRT31/KRT34-silenced ORS cell culture, even
if there was no distinct difference in TGFβ2-mediated ORS cell apoptosis. Finally, it was found that the
formation of anagen hair follicles and hair growth are suppressed in temporally KRT31/KRT34 silenced
mice, which could be recovered by intradermal injection of additional exogenous keratin.
Taken together, the results presented in this study reveal that hair regeneration is regulated by keratin-
mediated germ formation and DP condensation through biological events, including TGFβ2-induced ORS
cell apoptosis, caspase-mediated degradation of keratin, and spatial release and deposition of keratin
from the apoptotic ORS cells. Our pilot study indicate that keratin is not only a major structural
component of hair but can also play a functional role in the induction of HG formation and DP
condensation, facilitating entry into a new hair cycle. However, the precise identication of our proposed
cellular function of keratin in hair growth using a proper xenograft model is necessary, which is going on
as continuous study. In conclusion, considering the biological function of keratin in hair growth, our study
suggests that keratin can be a potent biomaterial for developing therapeutic agents for hair loss
treatment, and understanding how cellular behavior is regulated by spatiotemporal keratin release and
deposition from apoptotic epithelial cells can provide additional insight into deciphering cellular
interactions between epithelial cells and mesenchyme in the morphogenesis of other tissues.
Methods
Experimental Design
The aim of this study was to understand the biological function of keratin in hair growth. Firstly, to
determine the activity of hair growth, back-skin hairs in mice were removed, and hair follicle formation
and hair regrowth was evaluated after intradermal injection of hair-derived keratin (Fig. 1A-F). Next, to
dene cellular interaction of keratin with major cells participating in hair growth, cellular behavior such as
DP cell condensation and germ formation was studied by treating keratin in
in vitro
DP cell and ORS cell
culture (Fig. 1G-H, Fig. 2 and Fig. 3). From the results (Fig. 1-3), it was hypothesized that keratin-induced
hair growth could be closely related to a biological cascade happened during hair cycle with regards to
the exposure of keratin from TGF-b2-induced apoptotic ORS cells at stages of anagen-catagen transition.
To characterize keratin release or deposit from apoptotic ORS cells, apoptosis of ORS cells and its
following keratin release and deposit from TGF-b2-treated ORS cells were evaluated, and then DP cell
condensation was observed by the direct co-culture with TGF-b2-treated ORS cells and the DP cell culture
in conditioned medium collected from TGF-b2-treated ORS cell culture to evaluate the effect of released
or deposited keratin from apoptotic ORS cells on DP cell condensation (Fig. 4). Following DP cell
condensation induced by released and deposited keratin from apoptotic ORS cells, apoptosis-related
caspase 3 and caspase 6 expressions and caspase-mediated keratin degradation were characterized, and
the release and deposit of keratin through caspase-mediated keratin degradation in TGF-b2-treated
apoptotic ORS cells and its effect on DP cell condensation were evaluated by
in vitro
siRNA-mediated
silencing of caspase 6 mRNA expression in TGF-b2-treated apoptotic ORS cells (Fig. 5). To conrm the
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effect of keratin released from TGF-b2-treated apoptotic ORS cells on DP cell condensation, the keratin
were eliminated from conditioned medium collected from TGF-b2-treated cell culture by
immunodepletion, and DP cell condensation was evaluated with DP cell culture in the keratin-eliminating
conditioned medium collected from TGF-b2-treated apoptotic ORS cells (Fig. 6A-C). In company with
proving the effect of keratin released from TGF-b2-treated apoptotic ORS cells on DP cell condensation,
mRNA expressions of keratin 31 (KRT31) and keratin 34 (KRT34) were silenced in ORS cells by
in vitro
KRT31/KRT34 siRNA transfection, and then P-cadherin expressing germ formation of ORS cells was
observed in KRT31/KRT34-silenced ORS cell culture in the presence of TGF-b2 to evaluate the effect of
keratin spatially deposited from TGF-b2-treated apoptotic ORS cells on germ formation (Fig. 6D). Finally,
to study the role of keratin in hair follicle formation and hair growth
in vivo
, KRT31/KRT34 silencing was
processed by Invivofectamine KRT31/KRT34 siRNA transfection to mice (Fig. 7).
Cell Culture
Human outer root sheath cells (ORS; CEFO, CB-ORS-001) and human dermal papilla cells (DP; CEFO, CB-
HDP-001) were purchased and expanded in each human outer root sheath cell growth medium (CEFO, CB-
ORS-GM) and human dermal papilla growth medium (CEFO, CB-HDP-GM) at 37oC in a humidied
atmosphere containing 5 % CO2 according to the manufacturer's instructions. Cultures were fed every two
days and passaged by treatment with 0.25 % trypsin/EDTA (Gibco, 25200056), and the expanded DP
cells within 5 passages and ORS cells within 3 passages were used in this study.
Human Hair Keratin Extraction
Human hair keratin was extracted by slightly modied Sindai method, as reported previously 12, and
kindly provided by Gapi Bio. Detail methods of microwell fabrication are available inSupplementary
Methods.
Human Hair Keratin-mediated Hair Growth Test in Mice
For
in vivo
studies, male C57BL/6 mice were used, which were purchased from YoungBio (Samtako,
1404957265). The mice were housed under controlled condition at a temperature of 23 ± 2°C, humidity of
50 ± 5%, and light-dark cycle of 12 h. Mice were provided with a laboratory diet and water
ad libitum
. All
animal experiments were approved by the Institutional Animal Care and Use Committee of Konkuk
University (KU18159, KU19066), and procedures on animals were performed in accordance with the
relevant guidelines and regulations. The hair on the dorsal skin of mice was shaved repeatedly using an
electric clipper to synchronize the hair follicle cycle. Before treatment, the dorsal hair was completely
removed using the commercial hair removal cream Veet® (Reckitt Benckiser, 62200809951).
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To examine the hair growth promoting effect of keratin, 1.0 (w/v)% keratin in phosphate buffered saline
(PBS; Gibco, 10010023) was used. 10-week old mice were shaved repeatedly to synchronize the hair cycle
and randomly assigned to three groups: Neg. Con group; 3% Minoxidil group with daily topical application
of 100 μL of 3% Minoxidil (Minoxyl® 3%; Hyundai Pharm, Co., Seoul, Korea); 1.0 (w/v)% Keratin group
with intradermal injection of 100 μL of 1.0 (w/v)% keratin once. The mice were sacriced after 2 weeks.
Detail methods of the hair growth promoting effect of keratin according to keratin concentration are
available inSupplementary Methods.
Interaction Assay of DP Cells with Keratin
DP cells were seeded at a density of 2x104 cell/cm2 on 12 well and 6 well non-treated tissue culture plate
(SPL LIFE SCIENCES, 32012, 32006). The DP cells were adjusted to be stable for 1 day in human dermal
papilla growth medium (CEFO, CB-HDP-GM) at 37oC in a humidied atmosphere containing 5 % CO2 prior
to keratin treatment. After 1 day of adjustment, DP cells were cultured in human dermal papilla growth
medium containing 1.0(w/v)% keratin or not. The morphological change of DP cells in the presence of
keratin was observed under inverted uorescent microscopy (Olympus IX71), and the number of
condensed DP cell aggregates was counted. Cell proliferation upon keratin treatment was measured
using Cell Counting Kit-8 (Dojindo Molecular Technologies, CK04-20). DP cells were seeded on 12 well
non-treated tissue culture plate (SPL LIFE SCIENCES, 32012) at a seeding density of 1 × 104 cells/cm2,
and cultured in human dermal papilla growth medium (CEFO, CB-HDP-GM) containing 1.0(w/v)% keratin
or not in a humidied atmosphere of 5% CO2 at 37°C, and the medium was refreshed every two days. At
specic time points (1, 3 and 5 days), each well had 10 μL of the Cell Counting Kit-8 solution added and
then was incubated at 37°C for 2 h. Cell proliferation assays were performed in a 96-well plate reader by
measuring the absorbance at a wavelength of 450 nm.
For DP cell condensation assay according to different cell seeding density, DP cells were seeded at a
seeding density of 5x103 cell/cm2, 1x104 cell/cm2 and 2x104 cell/cm2 on 6 well non-treated tissue culture
plate (SPL LIFE SCIENCES, 32006). The DP cells were adjusted to be stable for 1 day in human dermal
papilla growth medium (CEFO, CB-HDP-GM) at 37oC in a humidied atmosphere containing 5 % CO2 prior
to keratin treatment. After 1 day of adjustment, DP cells were cultured in human dermal papilla growth
medium containing 1.0(w/v)% keratin or not. The number of condensed DP cell aggregates was counted
using inverted uorescent microscopy (Olympus IX71).
Detail method of interaction assay of DP cells with keratin on Matrigel is available inSupplementary
Methods.
Interaction Assay of ORS Cells with Keratin
Page 12/18
ORS cells were seeded at 2x104 cell/cm2 on 12 well and 6 well tissue culture plate (SPL LIFE SCIENCES,
30012, 30006). The ORS cells were adjusted to be stable for 1 day in human outer root sheath cell growth
medium (CEFO, CB-ORS-GM) at 37oC in a humidied atmosphere containing 5 % CO2 prior to keratin
treatment. After 1 day of adjustment, ORS cells were cultured in human dermal papilla growth medium
containing 1.0(w/v)% keratin or not. The morphological change of ORS cells in the presence of keratin
was observed under inverted uorescent microscopy (Olympus IX71) and time-lapse images were
captured. Cell proliferation upon keratin treatment was measured using Cell Counting Kit-8 (Dojindo
Molecular Technologies, CK04-20). ORS cells were seeded on 12 well tissue culture plate (SPL LIFE
SCIENCES, 30012) at a seeding density of 1 × 104 cells/cm2, and cultured in human outer root sheath cell
growth medium (CEFO, CB-ORS-GM) containing 1.0(w/v)% keratin or not in a humidied atmosphere of
5% CO2 at 37°C, and the medium was refreshed every two days. At specic time points (1, 3 and 5 days),
each well had 10 μL of the Cell Counting Kit-8 solution added and then was incubated at 37°C for 2 h. Cell
proliferation assays were performed in a 96-well plate reader by measuring the absorbance at a
wavelength of 450 nm.
RNA Extraction and Sequencing
To perform transcriptome sequencing (RNA-Seq) analysis of DP cells and ORS cells, total RNA was
extracted from the ORS and DP cells in the absences of keratin and in the presence of keratin. Detail
methods of RNA extraction, sequencing and differential gene expression analysis are available
inSupplementary Methods.
DP Cell Spheroid Formation and Maintenance assay of the
replated DP Cell Spheroids
For DP cell spheroid formation, cell spheroids as a micro tissue unit were generated by docking DP cells
into polyethylene glycol (PEG) microwell array with 450 µm in diameter. PEG microwells were fabricated
by microfabrication procedures, reported previously30. Detail methods of DP cell spheroid formation and
maintenance assay are available inSupplementary Methods.
TGFb2-mediated ORS Cell Apoptosis and Co-culture with DP Cells
ORS cells were seeded at 2x105 cell/cm2 on 12 well tissue culture plate (SPL LIFE SCIENCES, 30012) to
make conuent ORS cell layer. The ORS cells were adjusted to be stable for 1 day in human outer root
sheath cell growth medium (CEFO, CB-ORS-GM) at 37oC in a humidied atmosphere containing 5 % CO2.
After 1 day of adjustment, ORS cells were cultured in human dermal papilla growth medium containing
100ng/ml TGFb2 (PeproTech, 100-35B) for 5 days, and the media was refreshed every day. Detail
methods of DP cell condensation in direct co-culture of DP cells and TGFb2-treated ORS cells, and under
conditioned media from TGFb2-treated ORS cell layer are available inSupplementary Methods.
Page 13/18
Immunodepletion Study
To study the role of keratin released from TGFb2-induced apoptotic ORS cells in DP condensation and
germ formation of ORS cells, the released keratin in conditioned media from TGFb2-treated ORS cell layer
culture was removed by immunodepletion method. First, antibodies-conjugated beads were prepared as
follows; 150ml of nProtein A Sepharose (GE Healthcare, 17528001) was incubated with 400ml of guinea
pig anti-Type I+II Hair Keratins antibody (PROGEN, GP-panHK) or guinea pig normal IgG (Sigma-Aldrich,
I4756), as another negative control, for 18 h at 4°C. Non-specic binding was prevented with blocking
buffer containing 1% bovine serum albumin (BSA; Sigma-Aldrich, A9418) in TBS (Tris-Buffered Saline;
Biosesang, TR2005-000-74) with 0.1% Tween 20 (Duchefa Biochemie, P1362.1000) for 3 h at 4°C. The
conditioned media were collected from TGFb2-treated ORS cell layer cultured for 5 days, and 30ml of the
conditioned media were mixed with 75ml antibodies-conjugated beads, and then incubated with gentle
shaking overnight at 4°C. After incubation, antibodies-conjugated beads were removed by passing the
mixture through a Centrifuge Columns (Thermo Scientic, 89898). Detail methods of DP cell
condensation and P-cadherin expressing germ formation of ORS cells under keratin-removed conditioned
media are available inSupplementary Methods.
Caspase-3 and Caspase-6-mediated Hair Keratin Digestion
Assay
1(w/v)% hair keratin was dissolved in the reaction solution composing of 50mM HEPES (Gibco, 15630-
080), 50mM NaCl (JUNSEI CHEMICAL, 19015-1250), 0.1% CHAPS (Sigma-Aldrich, C3023), 10mM EDTA
(Sigma-Aldrich, 03609), 5% glycerol (SAMCHUN CHEMICALS, G0274) and 10mM DTT (Sigma-Aldrich,
43815) at pH 7.2. 5U/ml Casase-3 (Enzo, ALX-201-059) or 5U/ml Casase-6 (Enzo, ALX-201-060) was
added to the reaction solution containing hair keratin and incubated at 37oC for 0, 1, 3 and 24 hrs. After
the reaction, samples were denatured on 70°C for 10 min in LDS sample buffer (Invitrogen, B0007). Equal
amounts of denatured samples were loaded in pre-casted 4-12% Bis-Tris Plus Gels (Invitrogen,
NW04120BOX), and the electrophoresis was done by running at 200 V for 22 min. The gel was rinsed
three times with distilled water for 5 min each and stained by SimplyBlue SafeStain (Invitrogen, LC6060).
After 1 hr of staining, the gel was rinsed using distilled water until the background was removed
thoroughly, and then images of the gel was obtained using a commercialized scanner (Canon, TS8090).
In Vitro
Caspase-6 Gene Silencing Study
To evaluate the effect of caspase-6 mediated keratin degradation during TGFb2-induced ORS cell
apoptosis on keratin release or deposition and DP condensation, caspase-6 gene expression in ORS cells
was silenced by caspase-6 siRNA transfection. Detail methods of
in vitro
caspase-6 gene silencing study
are available inSupplementary Methods.
Page 14/18
In Vitro
KRT31/KRT34 Gene Silencing Study
To evaluate the effect of KRT31/KRT34 gene silencing during TGFb2-induced ORS cell apoptosis on
keratin release or deposition and germ formation of ORS cells, KRT31 and KRT34 gene expressions in
ORS cells were silenced by KRT31/KRT34 siRNA transfection. Detail methods of
in vitro
KRT31/KRT34
gene silencing study are available inSupplementary Methods.
Apoptosis and Growth Factor Antibody Array
TGFb2-induced ORS cell apoptosis was evaluated by comparative analysis using human apoptosis
antibody array (Abcam, ab134001), and the comparative analysis of growth factors present in the
conditioned medium collected from TGFb2-treated ORS cell culture in immunodepletion study were done
using human growth factor antibody array (Abcam, ab134002) according to manufacturer’s instructions.
Detail methods of apotosis array and growth factor antibody array analysis are available
inSupplementary Methods.
Western Blot Analysis
Molecular expressions of KRT34 and b-catenin in hair keratin-treated ORS cells, keratin content at protein
level in conditioned medium collected from TGFb2-treated ORS cell culture, the keratin content in keratin-
removed condition medium collected from TGFb2-treated ORS cell culture in immunodepletion study,
keratin content at protein level in conditioned medium collected from KRT31/KRT34-silenced ORS cell
culture or molecular keratin expression in KRT31/KRT34-silenced ORS cell, molecular expressions of
caspase 6 in TGFb2-treated ORS cells and keratin content at protein level in conditioned medium
collected from caspase 6-silenced ORS cell culture were evaluated by western blot analysis. Detail
method of western blot analysis is available inSupplementary Methods.
Real Time Quantitative Polymerase Chain Reaction (RT-
qPCR)
The gene expressions indicative of DP cell’s intrinsic property and TGFb2 gene expressions of DP cell
spheroids and the replated DP cell spheroids were evaluated by RT-qPCR. Detail method of RT-qPCR
analysis is available inSupplementary Methods.
Indirect Enzyme-Linked Immuno-Sorbent Assay (ELISA)
The molecular expressions indicative of DP cell’s intrinsic property from the replated DP spheroids
cultured in the presence of keratin were evaluated by ELISA. Detail method of ELISA is available
Page 15/18
inSupplementary Methods.
In Vivo
KRT31/KRT34 Gene Silencing Study
To conrm the effect of keratin on hair growth, KRT31/KRT34 were silenced by lipofectamine-mediated
delivery of KRT31/KRT34 siRNAs. First, Invivofectamine complex for KRT31/KRT34 siRNA delivery was
prepared as follows; siRNAs of KRT31 (Bioneer, 16660-1), KRT34 (Bioneer, 16672-1) and negative control
(Bioneer, SN-1003) were purchased, and siRNAs of KRT31 and KRT34 were dissolved in RNase-free water
as each 24 mg/ml concentration respectively. The two solutions, KRT31 siRNA and KRT34 siRNA, were
combined as 1:1 volume ratio to be 12 mg/ml of nal concentration. 12 mg of negative control siRNA
was also dissolved in 1 ml of RNase-free water. siRNAs-Invivofectamine (Thermo Fisher Scientic,
IVF3005) complex was prepared by the manufacturer's instruction, and 0.5 mg/ml of complex was
prepared nally prior to injection to mice. For in vivo study, 6-week-old mice were shaved repeatedly to
synchronize the hair cycle and randomly assigned to three groups: Con group with IV injection of 200 μl
of negative control siRNA injection; siRNA group with IV injection of 200 μL of KRT31/KRT34 siRNA
injection; siRNA+Keratin group with KRT31/KRT34 siRNA injection (IV, 200 μl) and intradermal injection of
total 100 μl of keratin a day after rst siRNA injection. For each group, mice were sacriced at either day 7
or day 14. Pictures of the back skin were taken at day 3, 7, 10, and 14 to examine the hair growth. The
silencing of KRT31/KRT34 gene expressions was conrmed by RT-qPCR. Detail method of RT-qPCR
analysis is available inSupplementary Methods..
Histological Analysis
The skin tissues were xed with 10% neutral-buffered formalin (BBC Biochemical, 0141). The tissues
were embedded in paran and sectioned at 4 μm thickness, followed by staining with hematoxylin and
eosin for histological analysis. The number of hair follicles in each cycle and diameter of anagen hair
follicles were quantied in multiple elds on perpendicular sections at ×100 magnication.
Immunocytochemical and Immunohistochemical Staining
Detail methods of immunocytochemical and immunohistochemical staining are available
inSupplementary Methods.
Statistical Analysis
All values obtained from
in vitro
and
in vivo
analysis are presented as the mean ± standard deviation
(SD). Statistically signicant differences were identied by two-sided Student’s t-test or one-way ANOVA
parametric test. A P-value of less than 0.05 was considered signicant.
Page 16/18
Declarations
Data Availability
The transcriptome sequencing data (RNA-Seq) have been deposited at NCBI GenBank under BioProject ID
PRJNA576064 (BioSample SAMN12924151 - SAMN12924158), and data are available in the private
reviewer link,https://dataview.ncbi.nlm.nih.gov/object/PRJNA576064?
reviewer=foebp22iqlsa9tg7mvrerhdbm6
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea
government (MSIT) (NRF-2016R1D1A1B03931933) and by the Bio & Medical Technology Development
Program of NRF funded by the Korean government (MSIT) (No. 2017M3A9E4048170), and by a fund
(2018ER610300) by Research of Korea Centers for Disease Control and Prevention.
Author Contributions
S.Y.A. performed most of the experiments and wrote the paper. S.Y.K. extract and puried human hair-
derived keratin. S.Y.V. extract and puried human hair-derived keratin and analyzed gene expressions
using real time-qPCR. E.J.C. carried out
in vivo
silencing experiment and histological analysis. H.J.K.
carried out
in vivo
mouse experiment and histological analysis. J.H.L. carried out RNA sequencing and
data analysis. S.W.H. and I.K.K. discussed results of the experiments and commented on the manuscript.
C.K.L. carried out
in vivo
mouse experiment and histological analysis. Y.S.H. and S.H.D. directed the
project, and Y.S.H. drafted the manuscript with input from all authors.
Competing Interests
The authors declare that they have no conict of interest.
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