HtrA1 is serine protease which has been identified in the epidermis.1 Although the precise function of HtrA1 in the epidermis is still unclear, some reports have suggested that HtrA1 is involved in programmed cell death, apoptosis and anoikis.2,3
On the other hand, it is generally known that excess apoptosis caused by UVB exposure is involved in the accumulation of cell damage in the skin epidermis, and it adversely affects skin epidermis homeostasis that causes chronic cell inflammation and parakeratosis. However, the precise mechanism underlying how UVB induced apoptosis adversely affects epidermal alterations has not been elucidated. This study aims to clarify the behaviour of HtrA1 in the apoptosis of epidermal cells induced by UVB exposure. Furthermore, we evaluated the efficacy of Japanese maple extract, a potent inhibitor of HtrA1 we found through this study.
Materials and methods
Normal human keratinocytes (NHKs, Kurabo) were purchased from Kurabo, and were cultured in Humedia-KG2 medium (KG2, Kurabo)
UVB source and irradiation
The UVB source was a Toshiba fluorescent sunlamp (FL-20SE), which has an emission spectrum from 280 nm to 370 nm, peaking at 305 nm. The UVB irradiance was measured using a UV light meter UV-340 (Lutron).
Western blotting for HtrA1
Normal human epidermal keratinocytes (NHKs, Kurabo) were exposed to UVB. The supernatant were concentrated by spin column (Millopore), and resolved on SDS-PAGE. HtrA1 was detected by western blot analysis using a specific antibody (anti HTRA1/PRSS11, R&D systems).
Inhibition against proteolytic activity of HtrA1
Human recombinant HtrA1 (Recombinant Human HTRA1/PRSS11, R&D systems) was pre-incubated with each plant extract for 10 min. After the incubation, the substrate β-casein (Sigma) was added into the mixture and incubated for 30 min. The mixtures were resolved on SDS-PAGE and the gels stained with Coomassie Briulliant Blue R 250 (Wako Pure Chemical).
Inhibitory effect of Japanese maple extract on apoptosis
NHKs were treated with Japanese maple extract for 3 hours. Then, cells were exposed to UVB. The cells were cultured for 24 hours with Japanese maple extract.
Intracellular nucleosomes were quantified by commercial assay ELISA kits (Cell Death Detection ELISA, Roche).
Measurement of VEGF secretion
NHKs were treated with HtrA1 for 24 hours in the presence or absence of Japanese maple extract. The amount of VEGF was quantified by ELISA kits (Quantikine ELISA Human VEGF, R&D systems).
A clinical study was conducted on 10 healthy volunteers in a double-blind test. The test was performed with an independent Institutional Review Board approval and following the Declaration ofHelsinki Principles. Subjects or legal guardians signed a written informed consent before the start of each study. 2% Japanese maple extract gel and a placebo gel were topically applied to the cheek twice a day for 4 weeks.
The treated area was measured for skin parameters (surface spots, wrinkles, textures, pores, UV spots, porphyrins, brown spots and red areas) by using VISIA Evolution (Canfield Imaging Systems).
Statistical analyses were performed using a two-tail paired t-test. Differences are considered significant if P<0.05 (indicated by *; P<0.05, **; P<0.01).
Results and discussion
UVB-induced HtrA1 secretion in NHKs
It is well established that cell apoptosis is triggered by many types of stimuli. In particular, UVB is well known to be an effective irritant to induce cell apoptosis with impairment of skin cells.
Then, we investigated the effect of UVB on levels of protein in HtrA1 of NHKs. As shown in Figure 1, HtrA1 secretion was not detected in non-UVB irradiation condition (left). On the other hand, HtrA1 was remarkably elevated after UVB irradiation in NHKs (right). This result suggested the possibility that UVB-induced overexpression of HtrA1 triggered the reaction of cell apoptosis.
Inhibitory effect of Japanese maple extract on HtrA1 activity
Since the proteolytic activity of HtrA1 made the cell apoptosis reaction progressed in UVB exposed NHKs, it was thought that the inhibitor of HtrA1 activity should be beneficial for assuagement of excess cell apoptosis. Then we performed the development of a novel inhibitor for HtrA1 activity. From the screening on 71 sorts of plant extract, we found that Japanese maple extract effectively inhibited β-casein degradation due to HtrA1 (Fig 2).
Inhibitory effect of Japanese maple extract on apoptosis
The effect of Japanese maple extract on apoptosis induced by UVB irradiation was examined. The nucleosome index, which was indicator of apoptosis, was increased by UVB irradiation. The apoptosis index was suppressed by treatment of Japanese maple extract, in a dose dependent manner (Fig 3).
Identification of active component of Japanese maple extract
To clarify the main component of activity in Japanese maple extract, we examined the inhibitory effect of two components isolated from Japanese maple extract. Component 2 indicated potent inhibitory effect on HtrA1 activity (Fig 4).
To evaluate the effect of Japanese maple extract on skin trouble, we carried out the clinical test by using the analysis of VISIA during autumn and winter. The number of skin pores was significantly decreased by topical application of Japanese maple extract gel (Table 1). Moreover, the red areas were drastically increased in the placebo treated group at 4 weeks. The application of Japanese maple extract gel inhibited the accumulation of red areas (Table 1, Red areas).
Significance: * p < 0.05 (vs. 4 weeks after placebo)
HtrA1-induced VEGF secretion in NHKs
The accumulation of red spot is closelyrelated to excess angiogenesis in skin. Then we examined the anti-angiogenesis potency of Japanese maple extract. It was reported that the overabundance of VEGF in skin epidermis positively contributes to excess capillary blood vessel formation in skin dermis.4 On the other hand, we demonstrated that the excess production of HtrA1 was observed in NHKs exposed to UVB. Therefore, we examined the direct effect of proteolytic activity of HtrA1 on VEGF production in NHKs. As shown in Figure 5, the production of VEGF was significantly increased by treatment of HtrA1 in NHKs.
Inhibitory effect of Japanese maple extract on VEGF release
We showed that the Japanese maple extract possesses inhibitory activity for HtrA1. To confirm whether Japanese maple extract inhibits VEGF secretion from NHKs, we examined the suppressive effect of Japanese maple extract on VEGF secretion induced by HtrA1 exposure. As shown in Figure 6, Japanese maple extract significantly inhibited the secretion of VEGF from NHKs
In this study, we demonstrated that UVB irradiation stimulated the production of HtrA1 in NHKs. In addition, it was found that Japanese maple extract, which is an HtrA1 inhibitor, suppressed an excess apoptosis induced by UVB irradiation. These findings strongly suggest that HtrA1 is involved in the regulation of epidermal cell death as one of its functions.
It is reported that an excess cell death of basal keratinocytes is involved in the induction of skin inflammation.5 Apoptosis is important to remove non-functional cells from inflammatory tissues and maintain a homeostasis of the organism. However, it is also indicated that excess cell death is closely linked to the accumulation of skin damage. In the clinical study, we found that Japanese maple extract improved the number of skin pore and red areas. Keratinisation process of epidermal cells is strictly regulated by programmed cell death and its disruption causes parakeratosis.
Since parakeratosis is related to an enlargement of pores, it is suggested that regulation of HtrA1 activity by Japanese maple extract resulted in the improvement of epidermal turnover, and moreover caused the improvement of pores.
In general, it is well known that regional skin redness is related to excess angiogenesis. Besides, it was reported that excess angiogenesis was induced by VEGF following UVB in epidermis.6 In this study, HtrA1 directly stimulated the secretion of VEGF from keratinocytes. The Japanese maple extract, which is an inhibitor of HtrA1 activity, significantly suppressed VEGF production. Therefore, these results suggested that there is a possible new angiogenesis pathway which may be intermediated by HtrA1 trigged by UVB irradiation in skin epidermis.
These findings all strongly suggest that Japanese maple extract could be a novel natural agent to assuage skin troubles, especially in skin pores and accumulation of redness, via controlling of HtrA1 proteolytic activity
1. De Luca A, De Falco M, Severino A et al. Distribution of the serine protease HtrA1 in normal human tissues, J. Histochem Cytochem. 2003; 51(10): 1279-1284.
2. Clausen T, Kaiser M, Huber R, Ehrmann M. HTRA proteases: regulated proteolysis in protein quality control, Nature Reviews Molecular Cell Biology2011; 12: 152-162.
3. Chien J, Aletti G, Baldi A, et al. Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity,J. Clin. Invest.2006; 116(7): 1994– 2004.
4. Yano K, Kadoya K, Kajiya K, Hong YK, Detmar M. Ultraviolet B irradiation of human skin induces an angiogenic switch that is mediated by upregulation of vascular endothelial growth factor and by downregulation of thrombospondin-1, Br J Dermatol.2005; 152 (1): 115-121.
5. Sato et al. Jpn. J. Dermatol.1997; 107(12): 1459-1471.
6. www.shiseidogroup.jp/rd/uptodate/sc2004_ 02.html
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