Biomolecules & Therapeutics 2023; 31(5): 550-558
Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth
Hae Chan Ha1,†, Dan Zhou1,2,†, Zhicheng Fu1,2, Moon Jung Back1, Ji Min Jang1,2, In Chul Shin1,2 and Dae Kyong Kim1,2,*
1Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, Seoul 06974,
2HaplnScience Research Institute, HaplnScience Inc., Seongnam 13494, Republic of Korea
Tel: +82-31-724-2611, Fax: +82-31-724-2612
The first two authors contributed equally to this work.
Received: April 4, 2023; Revised: June 5, 2023; Accepted: June 23, 2023; Published online: August 8, 2023.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Hair loss is a common condition that can have a negative impact on an individual’s quality of life. The severe side effects and the low efficacy of current hair loss medications create unmet needs in the field of hair loss treatment. Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1), one of the components of the extracellular matrix, has been shown to play a role in maintaining its integrity. HAPLN1 was examined for its ability to impact hair growth with less side effects than existing hair loss treatments. HAPLN1 was predominantly expressed in the anagen phase in three stages of the hair growth cycle in mice and promotes the proliferation of human hair matrix cells. Also, recombinant human HAPLN1 (rhHAPLN1) was shown to selectively increase the levels of transforming growth factor-β receptor II in human hair matrix cells. Furthermore, we observed concomitant activation of the ERK1/2 signaling pathway following treatment with rhHAPLN1. Our results indicate that rhHAPLN1 elicits its cell proliferation effect via the TGF-β2-induced ERK1/2 pathway. The prompt entering of the hair follicles into the anagen phase was observed in the rhHAPLN1-treated group, compared to the vehicle-treated group. Insights into the mechanism underlying such hair growth effects of HAPLN1 will provide a novel potential strategy for treating hair loss with much lower side effects than the current treatments.
Keywords: HAPLN1, Hyaluronic Acid, CD44, TGF-β receptor II, ERK1/2, HHGMCs

An estimated 147 million people suffer from hair loss worldwide, with an estimated 50% of men and women experiencing forms of pattern baldness at some point in their lives. Hair serves many features, providing insulation, protection, and a friction buffer; however, the aesthetic function of hair is arguably the most important for human beings because it is directly related to our sociality and self-esteem. Common treatments for hair loss include conventional chemical methods such as minoxidil, finasteride, herbal extracts, platelet-rich plasma (PRP), keratinocyte-conditioned media, adipose-derived stem cells, and hair transplantation. However, none of these methods are known to bring satisfactory results (Taghiabadi et al., 2020).

The hair follicle is an organ composed of inner and outer layers, bulge, bulb, and sebaceous glands (Philpott et al., 1990; Hibino and Nishiyama, 2004). The hair follicles undergo a hair growth cycle. Through the growing (anagen), degenerating (catagen), and resting periods (telogen), the hair repeats growth and loss (Chase, 1954). The anagen phase, the development process of dermal papilla (DP) and matrix cells, plays a crucial role in hair formation. Dermal papilla has blood vessels and nourishes matrix cells. In the matrix, hair is formed through the differentiation, proliferation, and keratinization of cells. The thickness and length of the hair are determined by the condition of dermal papilla and matrix cells. Several signaling molecules are involved in the normal hair follicle cycle, including Wnt, BMP, shh, and TGF-β signaling cascades (Niimori et al., 2012). TGF-β1 is known to inhibit the proliferation of keratinocytes (Mori et al., 1996; Foitzik et al., 2000). In a previous study, the onset of catagen in TGF-β1 -/- mice was slower than in the control group. While an injection of TGF-β1 into the dorsal skin of the mice induced catagen development (Foitzik et al., 2000). In contrast, TGF-β2 signal plays an important role in hair follicle morphogenesis and hair follicle stem cell activation (Hibino and Nishiyama, 2004; Oshimori and Fuchs, 2012). This notion is further supported by animal studies in which Tgfb2-null skin exhibited a delay in hair follicle morphogenesis (Foitzik et al., 1999).

TGF-β signaling contains two pathways: Smad-dependent or non-Smad-dependent pathways. In the canonical TGF-β pathway, TGF-β ligands bind to the TGF-β receptor II (TβRII) homodimers on the membrane with high affinity. TβRII dimerizes with TGF-β receptor I (TβRI) homodimers and activates TβRI by TβRII-mediated phosphorylation. The TβRI-TβRII complex undergoes either clathrin-mediated (non-raft) or caveolae-mediated (lipid-raft) endocytic pathway, and each pathway is determined by membrane trafficking of the TβRI-TβRII complex. Internalization of the TβRI-TβRII complex through clathrin-mediated endocytosis activates Smad2/3 pathway, and the internalized receptors can be recycled and return to the membrane. In contrast, caveolae-mediated endocytosis induces degradation of TβRI-TβRII complex (Huang and Chen, 2012). In the non-canonical pathway, TGF-β activates Smad-independent pathways such as PI3K/Akt, and MAPK pathway (Derynck and Zhang, 2003; Neuzillet et al., 2013). MAPKs, comprising JNK, p38, and ERK, are considered to play crucial roles in hair follicle morphogenesis and regeneration (Tang et al., 2019).

In the present study, we focus on the roles of Hyaluronan and proteoglycan link protein 1 (HAPLN1) in the hair cycle. HAPLN1, previously referred to as a link protein, is a glycoprotein that is distributed in various tissue organs such as cartilage, skin, brain, heart, and kidney and stabilizes non-covalent interactions between aggrecan and hyaluronic acid (HA) in proteoglycans (PGs) aggregates (Hardingham, 1979; Binette et al., 1994; Spicer et al., 2003). HA is a simple repeating disaccharide polymer, mainly found in the dermis and epidermis (50% of total body HA contents) (Papakonstantinou et al., 2012). One of the main characteristics of HA is its ability to hold water molecules, so it plays a vital role in keeping the skin hydrated and preventing the aging of skin and hair (Xi et al., 2019). As the ECM gradually degrades with age, HA also degrades along with it (Campiche et al., 2019). But HAPLN1 can retard the degradation of HA. A previous study has shown that HA levels were dramatically reduced in HAPLN1 knockdown, suggesting that loss of HAPLN1 protein affects the stability of HA (Govindan and Iovine, 2014). In hair, some studies have shown that HA promoted hair growth by promoting follicle growth (Kim et al., 2022).

In our previous study, using surgically anastomosed parabiotic mice and the aptamer-based proteomic analysis of their blood proteins, we identified HAPLN1 as a protein associated with skin aging. We have evaluated whether age-related decreases in the levels of collagen and HA, major structural components of the ECM, can be restored by exposure to youthful circulation (Fu et al., Hyaluronan and proteoglycan link 1- a novel signaling molecule for rejuvenating aged skin).

In the present study, we investigated the anti-aging effects of HAPLN1 on hair follicles. We focused on the proliferation of human hair germinal matrix cells (HHGMCs) and the mechanism by which recombinant human HAPLN1 (rhHAPLN1) is involved in the in vitro cell proliferation and in vivo hair growth in old mice. Our results revealed that rhHAPLN1 enhanced TGF-β2 signaling in a non-canonical pathway via the selective increase in TGF-β receptor II (TβRII) in HHGMCs. Furthermore, the data showed that rhHAPLN1-treated group into the synchronized old mice increased the hair growth through the earlier entrance into the anagen phase of the hair follicles.



Recombinant human TGF-β2 was purchased from R&D Systems (302-B2-010/CF, Minneapolis, MN, USA). 4-Methylumbelliferone (4-MU) and hyaluronic acid (HA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Recombinant human HAPLN1 was purchased from Cusabio (Houston, TX, USA).

Cell culture

Human hair germinal matrix cells were purchased from ScienCell Research Laboratories (HHGMCs, 2410, Carlsbad, CA, USA) and maintained in mesenchymal stem cell medium (MSCM, 7510, ScienCell Research Laboratories) and 5 mL FBS, 1% mesenchymal stem cell growth supplement (MSCGS, 7552, ScienCell Research Laboratories), and 1% of penicillin-streptomycin (ScienCell Research Laboratories) were grown at 37°C, 5% CO2. Primary HHGMCs between passages 2 and 6 were used in all experiments.

Western blotting

Western blot analysis was used to detect proteins in HHGMCs. Cells were lysed with RIPA buffer (BioWorld, Dublin, OH, USA) containing complete protease inhibitors (Roche, Mannheim, Germany) and phosphatase inhibitors (Roche), and the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA) was used for protein quantification. The protein samples were subsequently electrophoresed on 10% and 15% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked with 5% w/v skim milk/Bovine Serum Albumin (BSA) in TBST (TBS, 0.1% Tween-20) at room temperature for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-HAPLN1, anti-TβRI, anti-TβRII (Abcam, Cambridge, UK), anti-GAPDH, anti-MEK1 (Santa Cruz, Dallas, TX, USA), anti-c-Raf, anti-p-c-Raf, anti-ERK1/2, anti-p-ERK1/2, and anti-p-MEK1/2 (Cell Signaling, Denver, CO, USA). Following washing with TBST, the membranes were incubated with secondary antibody at room temperature for 1 h, washed with TBST and the resulting protein bands were visualized using ECL reagents (GE Healthcare, Buckinghamshire, UK) as a chemiluminescent substrate. The bands were analyzed and quantified using the computer software ImageJ (NIH, Bethesda, MD, USA).


HHGMCs were incubated in iced-cold solution of 0.25 mg/mL EZ-Link™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C for 1 h with gentle shaking on an orbital shaker. Then, 50 mM Tris-HCl (PH 7.5) was added to quench the reaction. The cells were harvested and sonicated with iced-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, phosphatase inhibitor and protease inhibitor). Pre-immobilized anti-biotin antibody-magnetic beads were treated overnight at 4°C. Bound proteins were released by boiling the anti-biotin antibody-magnetic beads with SDS-sample buffer, subjected to SDS-PAGE, followed by western blotting using the desired antibody.

siRNA and transfection

Small interference RNAs (siRNA) include siHAPLN1 (Dharmacon, Lafayette, CO, USA), negative control siRNA (siCTL; Dharmacon), Cluster of Differentiation 44 (CD44) siRNA (siCD44; Thermo Fisher Scientific), and negative control siRNA (siCTL; Thermo Fisher Scientific) were used at transfect to HHGMCs using Lipofectamine® RNAiMAX (Thermo Fisher Scientific) in low serum medium for 24 h. The cells were harvested 24 h after transfection.

Cell proliferation assay

A CCK-8 assay was used to determine proliferation according to the manufacturer’s protocol. HHGMCs were seeded in 96-well Poly-D-lysine coated plates (Corning® BioCoat™, Tewksbury, MA, USA) for 24 h. Then, 10 µL CCK-8 solution (Enzo Life Sciences, Farmingdale, NY, USA) was added to the well and incubated at 37°C for 1 h. After 1 h of incubation, the absorbance value at 450 nm of each well was measured using a microplate reader, after which the results were statistically analyzed.

Immunofluorescence staining

Immunofluorescence studies were performed using fresh frozen sections having a thickness of 8 µm. The sections were air dried for 1 h and fixed with 4% paraformaldehyde for 15 min at room temperature. After three washes with phosphate-buffered saline (PBS; Gibco, Carlsbad, CA, USA). Nonspecific sites were blocked with PBS with 0.5% normal goat serum at room temperature for 1 h and incubated with primary antibodies overnight at 4°C in a humidified chamber, and incubated with Alexa Fluor 488 or Alexa Fluor 594-conjugated to secondary antibodies (Invitrogen, Waltham, MA, USA) for 1 h at room temperature, followed by cell nucleus staining with DAPI for 10 min. The sections mounted with ProLong® Gold antifade reagent with DAPI (Thermo Fisher Scientific) and the staining results were observed microscopically.

Immunohistochemistry staining

Immunohistochemistry staining was performed using the recommended method of the instructions (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, USA). Eight micrometer-thick paraffin sections were deparaffinized and hydrated. The sections were boiled for 30 min in citrated buffer (10 mM citric acid, 0.05% tween 20, at PH 6.0) for antigen retrieval and cooled for 20 min at room temperature. Block endogenous peroxidase activity with H2O2. The sections blocked by 3% BSA in PBS at room temperature for 1 h, and incubated with primary antibodies overnight at 4°C in a humidified chamber. Sections were incubated with secondary antibodies for 1 h at room temperature, the ABC/DAB solution was added, and the sections were counterstained with hematoxylin. The sections were dehydrated in ethanol and the staining results were observed microscopically.

In-situ hybridization assay

In-situ hybridization (ISH) assay studies were performed using fresh frozen sections with a thickness of 11 μm. The cryosections were fixed in 4% paraformaldehyde at room temperature for 20 min and dehydrated with ethanol. The sections were incubated at room temperature for 10 min with H2O2 and incubation at 40°C for 30 min with a protease. HAPLN1 gene expression was detected with RNAScope 2.5 HD Reagent kit-brown (ACDbio, Newark, CA, USA). Briefly, HAPLN1 gene-specific probes were hybridized for 2 h at 40°C and a series of 6 amplification steps followed. Respectively, stringent washes with 1X wash buffers. A DAB-based chromogenic reagent was used to detect the brown signal for the HAPLN1 probe expression. Positive staining was indicated by brown granular dots present in the hair bulbs.


Pathogen-free, male C57BL/6NCrlOri mice (postnatal p23, p27, p32, p37, p40, p44) were purchased from Orient Bio Inc. (Seoul, Korea) and 20-month-old male C57BL/6J mice were purchased from Korea Basic Science Institute (Gwangju, Korea). Animals were quarantined on equal light/dark cycles (12/12 h) and were food and water ad libitum in a controlled environment at 22 ± 2°C and with a relative humidity of 50 ± 5%. All procedures and animal treatments were carried out in a clean room of the animal laboratory according to the guidelines for laboratory animal experimentation specified by Chung-Ang University (Seoul, Korea).

Study design

Progression to the anagen phase was induced by depilation of the skin on the dorsal aspect of mice. Therefore, we shaved the dorsal skin of 20-month-old male C57BL/6J mice (old mice) and hair follicles underwent one hair growth cycle for 4-6 weeks. To synchronize the hair growth cycle of old mice, this experiment was performed twice. rhHAPLN1 was dissolved in PBS at a concentration of 50 μg/mL and intraperitoneal injected at a dose of 0.1 mg/kg body weight. The old mice were randomized into two groups as follows: (i) the sham group or the vehicle-treated group received an intraperitoneal injection of PBS; (ii) the rhHAPLN1-treated group received HAPLN1 at a dose of 0.1 mg/kg body weight. The intraperitoneal injection of rhHAPLN1 was administered every three days for four weeks. The skin sample obtained four weeks after the last haircut. Hair coats were clipped and skin samples were embedded in the OCT (Sakura Finetek, Tokyo, Japan), frozen in liquid nitrogen and stored at –80°C. And paraffin section are skin samples fixed in formalin and embedded in paraffin. The protocol for animal care and use, observed in this study, was reviewed, and approved by the IACUC at Chung-Ang University (Approval number: 2017-00102).

Statistics analysis

All statistical analyses were performed using the GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). The data are presented as means ± standard error of the mean (SEM). Statistical significance was evaluated using a Student’s t-test and p-value <0.05 was considered statistically significant (*p<0.05, **p<0.001, ***p<0.0005, and ****p<0.0001).


HAPLN1 is expressed in hair follicles and promotes the proliferation of HHGMCs through TGFβ signaling

After examining HAPLN1 in the skin of young and old mice by immunohistochemistry staining, the results showed that HAPLN1 was located in hair follicles (yellow arrows) and skin fibroblasts (Fig. 1A). The level of HAPLN1 and the number of hair follicles were decreased in the skin sections of old mice. The mice hair follicles undergo hair growth cycle twice until postnatal 49 days. The first hair growth cycle is completed in about three and a half weeks (postnatal day 23–25) and the second hair growth cycle begins on postnatal 23 days and ends on postnatal 56 days (Sato et al., 1999). The time points for the second hair growth cycle are classified into different phases of the hair growth cycle based on established morphological guidelines as follow: early anagen (P23–25), mid anagen (P27), late anagen (P29–34), catagen (P37–39), and telogen (P42). The second telogen lasts more than two weeks, beginning around P42 (Alonso and Fuchs, 2006). To confirm how the hair growth stage affected HAPLN1, we stained P23, P32, P40, and P44 skin sections with hematoxylin and eosin (data not shown) and the levels of HAPLN1 were detected by immunofluorescence staining in three stages of the hair growth cycle (Fig. 1B). The level of HAPLN1 was significantly increased in the anagen phase, especially in hair bulbs. However, the level of HAPLN1 decreased in the catagen and telogen phases. To investigate HAPLN1 mRNA level, we compared the three stages of the hair growth cycle (Fig. 1C) by morphological criteria using in-situ hybridization staining. In the anagen phase, HAPLN1 mRNA was detected in hair bulbs (red arrows). These data show that HAPLN1 may be produced from the hair matrix cells and play an important role in their proliferation, leading to hair growth.

Figure 1. Expression of HAPLN1 indifferent stages of hair growth cycle and HAPLN1 enhanced the proliferation of HHGMCs. (A) Immunohistochemical staining of frozen sections of skin tissue. HAPLN1 (yellow arrows) staining of biopsies taken from 7-week-old mice and 20-month-old mice kept in conventional conditions (original magnifications: 100× and 200×; n=6). (B) Localization and characterization of HAPLN1 in the hair follicles of mice during each phase of the hair cycle. Localization of HAPLN1 in the hair follicles during anagen, catagen and telogen phases (original magnification, 400×; n=6). (C) In situ hybridization detection of HAPLN1 mRNA in each stage of hair growth cycle. The tissue sections were stained with HAPLN1 RNA probe according to the standard procedures (original magnification, 400×; n=6). (D) HHGMCs were pretreated with rhHAPLN (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h. The cell proliferation was analyzed with the CCK-8 assay (n=4). *p<0.05 versus corresponding TGF-β2 group. (E) Immunofluorescence staining of frozen sections of skin tissue. Localization of HAPLN1 in the hair growth cycle during anagen, catagen and telogen phases (original magnification, 400×; n=6).

To examine whether HAPLN1 contributes to hair cell proliferation, the HHGMCs were employed and incubated with rhHAPLN1 and/or HA, and the cell viability was analyzed with CCK-8 assay. The results show that the TGF-β2-dependent proliferation of HHGMCs was significantly promoted in the presence of rhHAPLN1 and/or HA (Fig. 1D). Co-localization of HAPLN1 and TβRII was detected in the hair matrix in the anagen phase (Fig. 1E). As confirmed in the skin section, HAPLN1 was detected in the hair matrix with a high rate of cell proliferation, suggesting that HAPLN1 may be an essential factor for cell proliferation.

rhHAPLN1 selectively enhanced the level of TβRII in HHGMCs

We examined the effect of rhHAPLN1 on the levels of TGF-β receptor I and II in HHGMCs using western blotting. rhHAPLN1 increased the level of TβRII at a concentration of 20 ng/mL (Fig. 2A), and significantly increased the TβRII levels when combined with HA (Fig. 2B). This suggests that such increases by rhHAPLN1 and/or HA may be due to the formation of a huge PGs-rhHAPLN1-HA-CD44-TβRII complex, as previously described (Bourguignon et al., 2002; Harada and Takahashi., 2007), thereby preventing the rate of endocytic degradation of the TβRII.

Figure 2. rhHAPLN1 and/or HA increased TβRII levels expression in HHGMCs. (A) The plot and relative quantification of the expression of TβRII in HHGMCs treated with various doses (0 ng/mL; 5 ng/mL; 10 ng/mL; 20 ng/mL) rhHAPLN1 for 24 h. (B) The plot and the relative quantification of the expression of TβRII in HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h. (C) The plot and the relative quantification of the expression of TβRII in HHGMCs following HAPLN1 knockdown. (D) The plot and relative quantification of the expression of TβRII in HAPLN1 knockdown HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h. (E) The plot and the relative quantification of the expression of TβRII and HAS2 in HHGMCs pretreated with 4-MU (0.5 mM) and/or rhHAPLN1 (25 ng/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h (n=3). *p<0.05, **p<0.01 versus corresponding control group (HA/HAPLN1 0 ng/mL) or siCTL group.

To examine whether TβRII levels are down-regulated by the knock-down of HAPLN1, HHGMCs were transfected with either siCTL or siHAPLN1. The siHAPLN1 reduced endogenous HAPLN1 and concurrent reduction of the TβRII levels (Fig. 2C). When siHAPLN1-transfected HHGMCs were treated with rhHAPLN1 and/or HA, the TβRII levels were significantly restored (Fig. 2D). Thus, we suggest that endogenous HAPLN1 may play an important role in enhancing or maintaining the TβRII level. On the other hand, to investigate whether rhHAPLN1 depends on HA to increase the TβRII levels, 4-MU, an inhibitor of hyaluronan synthase 2 (HAS2), was employed (Kultti et al., 2009). 4-MU significantly decreased TβRII and HAS2 expression levels, but rhHAPLN1 restored TβRII and HAS2 expression levels (Fig. 2E), suggesting that HA may restore the reduced TβRII level through rhHAPLN1.

CD44 is essential for rhHAPLN1 to increase the level of TβRII

It is well known that HA binds to the CD44 receptors, and these complexes seem to be further associated with TβRII at the cell surface, leading to activation of TGF-β signaling (Bourguignon et al., 2002). To evaluate the effect of CD44 knockdown on HAPLN1-regulated TβRII, HHGMCs were transfected with siCTL and siCD44. Our results showed that the knockdown of CD44 inhibited TβRII level (Fig. 3A). Moreover, following treatment with HA and/or rhHAPLN1 on siCD44-transfected HHGMCs, TβRII levels were not restored by HA and/or rhHAPLN1 (Fig. 3B). The results indicate that CD44 is an essential factor in the action of rhHAPLN1 on the regulation of TβRII level.

Figure 3. Lack of endogenous CD44 decreased TβRII levels expression in HHGMCs. (A) The plot and the relative quantification of the expression of TβRII in CD44 following HAPLN1 knockdown. (B) The plot and the relative quantification of the expression of TβRII in CD44 knockdown HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h (n=3). *p<0.05, **p<0.01 versus corresponding siCTL group.

rhHAPLN1 and HA increase the level of cell surface membrane TβRII by the treatment of TGF-β2 in HHGMCs

Previous findings suggested that HA and HAPLN1 exert their effects at the level of the cell membrane. This is because extracellular HA and HAPLN1 were found to regulate TβRII, and CD44 was found to play a crucial role in this process. In treatment with HA and/or rhHAPLN1 on HHGMCs, cell surface proteins were labeled with sulfo-NHS-LC-biotin and then separated using immunoprecipitation analysis. As per the results, the exogenous HA and rhHAPLN1 increased the level of TβRII at the cell surface membrane in HHGMCs (Fig. 4). These results imply that the increased levels of TβRII by rhHAPLN1 and/or HA may indeed be through a mechanism at the cell membrane.

Figure 4. rhHAPLN1 and/or HA increased TβRII levels expression in HHGMCs surface membrane. The plot and the relative quantification of the expression of TβRII in HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 1 h prior to stimulation with TGF-β2 (2 ng/mL) for 23 h. Cell surface levels of TβRII were defined by labeling cells with sulpho-NHS-LC-biotin. Immunoprecipitation study was performed using an antibody against biotin (n=3). **p<0.01 versus corresponding control group (HA/HAPLN1 0 ng/mL).

rhHAPLN1 and HA promote TGF-β2 signaling through the ERK1/2-mediated non-canonical pathway

It has been previously shown that the heteromeric receptor complexes between TβRI and TβRII mediated by TGF-β stimulate distinct downstream signaling pathways, Smad-dependent and Smad-independent pathways, also termed canonical and non-canonical pathways, respectively, to regulate different context-dependent transcriptions (Massagué et al., 2000). That is, the canonical TGF-β signaling pathway uses Smad2 and/or Smad3 to transfer signals. Smad2/3 are directly phosphorylated by TβRI and translocate to the nucleus to regulate gene transcription (Massagué et al., 2000). One of the non-canonical TGF-β signaling pathways involves ERK/MAPK kinase. The ERK/MAPK pathway is induced by various stimuli. Thus, we investigated whether the TGF-β2 signaling occurred through the Smad-dependent or Smad-independent pathway. No changes in phosphorylation of Smad2 was observed by the treatment of rhHAPLN1 and/or HA (Fig. 5A). In contrast, interestingly, p-c-Raf, p-MEK1/2, and p-ERK1/2, which are known as the effectors activated downstream of the Smad-independent pathway, were significantly increased in the presence of rhHAPLN1 and/or HA (Fig. 5B). These results highlight that the TGF-β2 signaling could undergo via the non-canonical ERK1/2 pathway.

Figure 5. rhHAPLN1 and/or HA activate non-canonical pathway of TGF-β2-ERK1/2 signaling. The plot and the relative quantification of the expression of phosphorylation of (A) Smad2, (B) c-Raf, MEK1/2 and ERK1/2 in HHGMCs pretreated with rhHAPLN1 (25 ng/mL) and/or HA (25 μg/mL) for 23 h prior to stimulation with TGF-β2 (2 ng/mL) for 1 h (n=3). **p<0.01, ***p<0.001 and ****p<0.0001 versus corresponding control group (HA/HAPLN1 0 ng/mL).

rhHAPLN1 stimulates hair growth in mice

Our previous results indicating the loss of HAPLN1 in the aging process, prompted us to adapt old mice for an in vivo experiment. Each of the mice has been shown to enter the different hair growth cycles after the second hair growth cycle. To synchronize the hair growth cycle, the old mice were clipped, and depilatory cream was applied to stimulate the whole skin. In old mice, hair follicles enter the anagen phase earlier in the rhHAPLN1-treated group compared to the vehicle-treated group (Fig. 6).

Figure 6. rhHAPLN1 promotes hair regrowth in mice. 20 month-old male C57BL/6 mice were depilated and were treated with rhHAPLN1, beginning the day after hair removal, three times per week. Photographs were taken on days 0, 8, and 21 days after depilation. From day 8, rhHAPLN1-treated group showed notable darkening of skin color, and over time they showed a markedly greater hair growth than the vehicle-treated group (n=6).

Hair loss in a part of the head has been known to be due to various causes, such as drugs, stresses, and autoimmune disorders. Various therapeutic agents have been shown to reverse it depending on the cause of the condition, as well as patient sex and age (Kim et al., 2022). This study is the first to describe the newly discovered effects of full-length rhHAPLN1 on hair growth of old mice as well as the underlying mechanisms in HHGMCs. Indeed, HAPLN1 has been shown to bind noncovalently to a specific region of proteoglycan protein and a filament of HA and stabilizes the reversible noncovalent binding between. Thus, HA-PGs complexes known as ‘aggregates’ are formed in the PCM (Hardingham, 1979; Buckwalter et al., 1984; Knudson and Knudson, 1993). It has long been known that the aggregate formed in the presence of HAPLN1, termed ‘HAPLN1-containing aggregate’ is five times longer and has three times more proteoglycans compared to HAPLN1-free aggregate because the former leads to an increased stability of the linking between proteoglycans and HA (Buckwalter et al., 1984). Thus, HAPLN1 can largely contribute to the formation of a water-rich and viscoelastic hydrogel matrix (Warren et al., 2021).

A previous study showed that the exogenous HA induced the trafficking of TβR to caveolin-1 lipid raft-associated pools and caused TβR degradation (Ito et al., 2004). Of note, the present study demonstrates that the treatment with rhHAPLN1 and/or HA selectively increased the levels of TβRII, not TβRI, at the cell surface membrane in HHGMCs. Although at present, the precise mechanisms by which rhHAPLN1 increased the levels of TβRII, rhHAPLN1 seems to inhibit the internalization of TβRII by preventing the degradation of HA (Govindan and Iovine, 2014). In this context, it should be noted that the degradation of HA is essential for the endocytosis of large HA molecules (Hua et al., 1993; Danielson et al., 2015), and this process involves CD44, the main receptor of HA (Harada and Takahashi, 2007). Since TβRs is known to be directly linked to CD44, and the degradation of HA is required for the endocytosis of TβR (Bourguignon et al., 2002; Ito et al., 2004). Additionally, as aforementioned, the HAPLN1-containing aggregate binds to CD44 as a bulky and water-rich hydrogel ligand, forming a huge size of complex consisted of the aggregate-CD44-TβRs, and thereby slowing down the rate of endocytic degradation of TβRII. Therefore, our data suggest that rhHAPLN1 could contribute to the increase in the levels of TβRII by preventing the degradation of HA, enabling the aggregate-containing complexes to remain functional on the membrane. Moreover, our results indicate that exogenous HA and/or rhHAPLN1 increase the level of cell surface membrane TβRII in the presence of TGF-β2, suggesting that rhHAPLN1 and/or HA may decrease the internalization rate of TβRII, and subsequently enhance the TGF-β2-signaling through such an increase in the number of TβRII on the cell surface membrane. Our results show that when membrane proteins are labeled with sulfo-NHS-LC-biotin, the increased levels of TβRII were shown in HA- and/or rhHAPLN1-treated HHGMCs through an immunoprecipitation analysis, further indicating the decreased rate of the endocytic degradation of TβRII.

Importantly, we found that both the HA and CD44 are likely to be essential for rhHAPLN1 to act on HHGMCs and regulate TGF-β2 signaling. When production of endogenous HA was inhibited by the HAS2 inhibitor 4-MU, the levels of TβRII were significantly decreased, suggesting an important role of HA as a molecule connecting between TβRII and rhHAPLN1. Furthermore, in CD44-knockdown HHGMCs, TβRII was not only decreased compared with the control siRNA (siCTL), but also unaffected by the treatment of HA and/or rhHAPLN1, highlighting the important roles of CD44 in the enhancement of TβRII by rhHAPLN1. Unlike young mice, old mice have been shown to exhibit different hair growth cycles for each individual (Plikus and Chuong, 2008). In this regard, old mice were clipped and depilatory cream was applied to stimulate the whole skin. The hair growth cycle was promoted by skin stimulation and the above procedure was repeated twice to synchronize the hair growth cycle of all old mice. After the second hair growth cycle, we clipped hair coats in the telogen phase and treated them with rhHAPLN1. Compared with the vehicle-treated group, the rhHAPLN1-treated group showed that the hair follicles entered the hair growth phase (anagen) earlier. rhHAPLN1 seems to play a role in optimizing an environment for cell proliferation and differentiation, allowing hair follicles to enter the anagen phase in vivo.

Taken together, the present study findings show that the treatment of rhHAPLN1 and/or HA can result in an increase in TβRII protein levels, thus leading to the enhancement of TGF-β2-ERK1/2 signaling via non-canonical TGF-β signaling pathways. In addition, these data imply that TβRII may be subjected to its degradation via an endocytic process, but this degradation pathway may be slowed down or inhibited by the mechanochemical properties of rhHAPLN1. Interestingly, the rhHAPLN1-treated group showed that the hair follicles entered the hair growth phase (anagen) earlier, allowing hair follicles to enter the anagen phase in vivo. Thus, rhHAPLN1 may act as a novel biomechanical signaling protein promoting hair growth.

This research was supported by the Chung-Ang University Young Scientist Scholarship in 2017, and also supported by a grant from the National Research Foundation of Korea (NRF-2017M3A9D8048414) funded by the Korean government (Ministry of Science and ICT).

D. Zhou, Z. Fu, JM. Jang, and IC. Shin are employees of HaplnScience Inc. JM. Jang and DK. Kim are shareholders of HaplnScience Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

  1. Alonso, L. and Fuchs, E. (2006) The hair cycle. J. Cell Sci. 119, 391-393.
    Pubmed CrossRef
  2. Binette, F., Cravens, J., Kahoussi, B., Haudenschild, D. R. and Goetinck, P. F. (1994) Link protein is ubiquitously expressed in non-cartilaginous tissues where it enhances and sta-bilizes the interaction of proteoglycans with hyaluronic acid. J. Biol. Chem. 269, 19116-19122.
    Pubmed CrossRef
  3. Bourguignon, L. Y., Singleton, P. A., Zhu, H. and Zhou, B. (2002) Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells. J. Biol. Chem. 277, 39703-39712.
    Pubmed CrossRef
  4. Buckwalter, J. A., Rosenberg, L. C. and Tang, L. H. (1984) The effect of link protein on proteoglycan aggregate structure. An electron microscopic study of the molecular architecture and dimensions of proteoglycan aggregates reassembled from the proteoglycan monomers and link proteins of bovine fetal epiphyseal cartilage. J. Biol. Chem. 259, 5361-5363.
    Pubmed CrossRef
  5. Chase, H. B. (1954) Growth of the hair. Physiol. Rev. 34, 113-126.
    Pubmed CrossRef
  6. Campiche, R., Jackson, E., Laurent, G., Roche, M., Gougeon, S., Séroul, P., Ströbel, S., Massironi, M. and Gempeler, M. (2019) Skin filling and firming activity of a hyaluronic acid inducing synthetic tripeptide. Int. J. Pept. Res. Ther. 26, 181-189.
  7. Derynck, R. and Zhang, Y. E. (2003) Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425, 577-584.
    Pubmed CrossRef
  8. Danielson, B. T., Knudson, C. B. and Knudson, W. (2015) Extracellular processing of the cartilage proteoglycan aggregate and its effect on CD44-mediated internalization of hyaluronan. J. Biol. Chem. 290, 9555-9570.
    Pubmed KoreaMed CrossRef
  9. Foitzik, K., Lindner, G., Mueller-Roever, S., Maurer, M., Botchkareva, N., Botchkarev, V., Handjiski, B., Metz, M., Hibino, T., Soma, T., Dotto, G. P. and Paus, R. (2000) Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo. FASEB J. 14, 752-760.
    Pubmed CrossRef
  10. Foitzik, K., Paus, R., Doetschman, T. and Dotto, G. P. (1999) The TGF-beta2 isoform is both a required and sufficient inducer of murine hair follicle morphogenesis. Dev. Biol. 212, 278-289.
    Pubmed CrossRef
  11. Govindan, J. and Iovine, M. K. (2014) Hapln1a is required for connexin43-dependent growth and patterning in the regenerating fin skeleton. PLoS One 9, e88574.
    Pubmed KoreaMed CrossRef
  12. Hibino, T. and Nishiyama, T. (2004) Role of TGF-beta2 in the human hair cycle. J. Dermatol. Sci. 35, 9-18.
    Pubmed CrossRef
  13. Huang, F. and Chen, Y. G. (2012) Regulation of TGF-beta receptor activity. Cell Biosci. 2, 9.
    Pubmed KoreaMed CrossRef
  14. Hardingham, T. E. (1979) The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem. J. 177, 237-247.
    Pubmed KoreaMed CrossRef
  15. Harada, H. and Takahashi, M. (2007) CD44-dependent intracellular and extracellular catabolism of hyaluronic acid by hyaluronidase-1 and -2. J. Biol. Chem. 282, 5597-5607.
    Pubmed CrossRef
  16. Hua, Q., Knudson, C. B. and Knudson (1993) Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis. J. Cell Sci. 106, 365-375.
    Pubmed CrossRef
  17. Ito, T., Williams, J. D., Fraser, D. J. and Phillips, A. O. (2004) Hyaluronan regulates transforming growth factor-beta1 receptor compartmentalization. J. Biol. Chem. 279, 25326-25332.
    Pubmed CrossRef
  18. Kim, M. J., Seong, K. Y., Kim, D. S., Jeong, J. S., Kim, S. Y., Lee, S., Yang, S. Y. and An, B. S. (2022) Minoxidil-loaded hyaluronic acid dissolving microneedles to alleviate hair loss in an alopecia animal model. Acta Biomater. 143, 189-202.
    Pubmed CrossRef
  19. Kultti, A., Pasonen-Seppanen, S., Jauhiainen, M., Rilla, K. J., Kärnä, R., Pyöriä, E., Tammi, R. H. and Tammi, M. I. (2009) 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3. Exp. Cell Res. 315, 1914-1923.
    Pubmed CrossRef
  20. Knudson, C. B. and Knudson, W. (1993) Hyaluronan-binding proteins in development, tissue homeostasis, and disease. FASEB J. 7, 1233-1241.
    Pubmed CrossRef
  21. Mori, O., Hachisuka, H. and Sasai, Y. (1996) Effects of transforming growth factor beta 1 in the hair cycle. J. Dermatol. 23, 89-94.
    Pubmed CrossRef
  22. Massagué, J., Blain, S. W. and Lo, R. S. (2000) TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 103, 295-309.
    Pubmed CrossRef
  23. Niimori, D., Kawano, R., Felemban, A., Niimori-Kita, K., Tanaka, H., Ihn, H. and Ohta, K. (2012) Tsukushi controls the hair cycle by regulating TGF-β1 signaling. Dev. Biol. 372, 81-87.
    Pubmed CrossRef
  24. Neuzillet, C., Hammel, P., Tijeras-Raballand, A., Couvelard, A. and Raymond, E. (2013) Targeting the Ras-ERK pathway in pancreatic adenocarcinoma. Cancer Metastasis Rev. 32, 147-162.
    Pubmed CrossRef
  25. Oshimori, N. and Fuchs, E. (2012) Paracrine TGF-beta signaling counterbalances BMP-mediated repression in hair follicle stem cell activation. Cell Stem Cell 10, 63-75.
    Pubmed KoreaMed CrossRef
  26. Philpott, M. P., Green, M. R. and Kealey, T. (1990) Human hair growth in vitro. J. Cell Sci. 97, 463-471.
    Pubmed CrossRef
  27. Papakonstantinou, E., Roth, M. and Karakiulakis, G. (2012) Hyaluronic acid: a key molecule in skin aging. Dermatoendocrinol 4, 253-258.
    Pubmed KoreaMed CrossRef
  28. Plikus, M. V. and Chuong, C. M. (2008) Complex hair cycle domain patterns and regenerative hair waves in living rodents. J. Invest. Dermatol. 128, 1071-1080.
    Pubmed KoreaMed CrossRef
  29. Spicer, A. P., Joo, A. and Bowling, R. A., Jr. (2003) A hyaluronan binding link protein gene family whose members are physically linked adjacent to chondroitin sulfate proteoglycan core protein genes: the missing links. J. Biol. Chem. 278, 21083-21091.
    Pubmed CrossRef
  30. Sato, N., Leopold, P. L. and Crystal, R. G. (1999) Induction of the hair growth phase in postnatal mice by localized transient expression of Sonic hedgehog. J. Clin. Investig. 104, 855-864.
    Pubmed KoreaMed CrossRef
  31. Taghiabadi, E., Nilforoushzadeh, M. A. and Aghdami, N. (2020) Maintaining hair inductivity in human dermal papilla cells: a review of effective methods. Skin Pharmacol. Physiol. 33, 280-292.
    Pubmed CrossRef
  32. Tang, P., Wang, X., Zhang, M., Huang, S., Lin, C., Yan, F., Deng, Y., Zhang, L. and Zhang, L. (2019) Activin B stimulates mouse vibrissae growth and regulates cell proliferation and cell cycle progression of hair matrix cells through ERK signaling. Int. J. Mol. Sci. 20, 853.
    Pubmed KoreaMed CrossRef
  33. Warren, J. P., Miles, D. E., Kapur, N., Wilcox, R. K. and Beales, P. A. (2021) Hydrodynamic mixing tunes the stiffness of proteoglycan-mimicking physical hydrogels. Adv. Healthc. Mater. 10, e2001998.
    Pubmed CrossRef
  34. Xi, J., Ting, Y. L., Xue, L. and Dan, L. (2019) Effect of hyaluronic acid: Mechanistic investigations via topological and functional analysis of its protein interaction network. Trop. J. Pharm. Res. 18, 1919-1925.

This Article

Cited By Articles
  • CrossRef (0)

Funding Information

Social Network Service