Royal Jelly Extract Accelerates Keratinocyte Proliferation, and Upregulates Laminin α 3 and Integrin β 1 mRNA Expression, via Akt/mTOR/HIF-1 α Pathway

Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.


Introduction
As a royal jelly, one of the bee products, is applied for alternative and modern medicine and cosmetics, pharmacological and biochemical studies on its benefits for health are being actively conducted. Approximately 185 organic compounds have been detected in royal jelly, which has various benefits and biological activities such as in reproductive health, neurodegenerative diseases, and tumor treatment [1] [2] [3] [4]. New evidence of the effect of royal jelly in anti-dermatitis [5] [6], wound healing [7], collagen production [8] and anti-melanogenesis [9] have been accumulated in the last two decades. Thus, royal jelly is considered an ideal cosmetics and skin care product component.
Epidermal basement membrane (EBM) is a sheet-like polymeric structure primarily composed of laminin, type IV collagen, perlecan and nidogen. Laminin is important for not only building the EBM framework but also for interaction with basal keratinocytes contributing to epidermis homeostasis. Although 15 laminin isoforms, composed of the combination of five distinct α subunits, three β subunits and three γ subunits, have been identified, the EBM is only enriched in laminin-332 (α3β3γ2) and laminin-511 (α5β1γ1) [10]. On the contrary, integrins are heterodimeric transmembrane receptors consisting of one α and one β subunit. α3β1 and α6β4 integrins are constitutively and abundantly expressed on the basal epidermal surface of basal keratinocytes [11] and interact with laminin-332 and laminin-511 [10] [12]. While α6β4 integrin is crucial for basal keratinocyte anchorage to the EBM, β1-containing integrins are involved in various cell functions after ligation to extracellular matrix molecules such as laminins [13]. Previous studies have reported that β1 integrin determines keratinocyte stem cell fate [14] and the interaction of laminin-332 and α3β1 integrin plays an indispensable role in epithelial cell proliferation [15].
Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcriptional factor, regulates about 200 genes involved in several cellular and systemic responses [16] [17]. HIF-1 activity basically depends on oxygen level, however, previous studies have demonstrated various mediators such as reactive oxygen species, cytokines, and growth factors [18] [19]. Moreover, HIF-1α level increases in the psoriatic lesions, and it is involved in keratinocyte proliferation [20] [21]. As the skin is originally hypoxic [22], HIF-1 may participate in biological and pathological processes in the skin. This study was designed to explore the effects of royal jelly extract (RJ) on keratinocyte proliferation, as well as determine its underlying molecular mechanism.

Treatment with RJ and Reagents
HaCaT cells were seeded into 24 and 96-well plates (1 × 10 5 and 2 × 10 3 cells/ well, respectively) for quantitative PCR (qPCR) and into bromodeoxyuridine (BrdU) uptake assay, respectively, and then maintained in a 5% CO 2 -humidified atmosphere at 37˚C. After cultivation for 24 h, the cells were treated with 2% RJ in the presence or absence of reagents for appropriate periods.

Cell Proliferation Assay
To estimate cell proliferation, BrdU cell proliferation ELISA kit (Abcam, Cambridge, UK) was used, according to the manufacturer's instruction.

RNA Isolation and qPCR
The treated and control cells were harvested and total RNA was extracted with SV RNA isolation kit (Promega; Madison, WI, USA), according to the manufacturer's instructions, followed by reverse transcription using ReverTra Ace ®

Immunoblotting
HaCaT cells were collected in the RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. Equal amount of protein (10 μg

Statistical Analysis
Results of mRNA relative expression is expressed as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analysis was performed using the Student's t-test. Statistical significance was set p < 0.05.

RJ Accelerates Cell Proliferation, and Enhances Laminin α3 and Integrin β1 Expression
The effect of RJ on keratinocyte proliferation was examined using BrdU uptake assay. The proliferation of RJ-treated cells was significantly accelerated to 149.6% ± 5.7% than that in the control (Figure 1(a)). Laminin α3 (LAMA3) and integrin β1 (ITGB1) mRNA expression levels were significantly upregulated to 1.46 ± 0.14 and 1.51 ± 0.21-fold, respectively (Figure 1(b) and Figure 1(c)).

LAMA3/ITGB1/ILK Pathway Is Not Involved in RJ-Induced Cell Proliferation Acceleration
The contribution of LAMA3, ITGB1 and ILK in RJ-induced cell proliferation acceleration was evaluated using LAMA3 and ILK silencing and antibody-mediated ITGB1 blocking. RJ accelerated the proliferation in both LAMA3 knockdown (KD), as well as mock treatment (MT) cells (Figure 2(a)). The ITGB1 blocking (ITGB1 BL) by pretreatment with 10 μg/ml anti-ITGB1 antibody did not affect the RJ-induced proliferation acceleration (Figure 2(b)). Moreover, the RJ-induced proliferation acceleration was retained in ILK KD cells (Figure 2(c)).

Akt and mTOR Inhibitors Suppress RJ-Induced Cell Proliferation Acceleration and HIF-1α Expression Upregulation
The effects of 10 μM GSK690693, an Akt inhibitor, and 2 μM rapamycin, an A. Aioi

Discussion
Since various benefits and biological activities of royal jelly have been reported [1]- [9], we explored the effects of RJ on keratinocyte proliferation and its underlying molecular mechanism in this study. RJ treatment accelerated keratinocyte proliferation and enhanced LAMA3 and ITGB1 mRNA expression levels ( Figure 1). LAMA3, a subunit comprising laminin-332, interacts with integrin α3β1 and α6β4 [11]. Previous studies have shown that LAMA3 ligation to ITGB1 affects epidermal stem cell fate and gap junctional communication in skin [23] [24]. Moreover, the interaction between laminin-332 and integrin α3β1, followed by mitogen-activated protein kinase activation regulates epithelial cell proliferation [15] and ILK is dispensable for epidermis construction [25] [26] [27]. Therefore, we first hypothesized that laminin-332, integrin α3β1, and ILK   [31]. As Akt and mTOR activate NF-κB through inducing IKK degradation by phosphorylation [32], we examined NF-κB activation by detecting phospho-NF-κB p65. RJ treatment induces NF-κB p65 phosphorylation ( Figure 5), suggesting that RJ activates NF-κB. Collectively, our results suggest that RJ upregulates LAMA3 and ITGB1 mRNA expression by the direct effect of HIF-1α expression enhancement and accelerates keratinocyte proliferation via Akt/mTOR/ HIF-1α/NF-κB signaling pathway ( Figure 6). Conclusively, since epidermal HIF-1α loss accelerates epidermal aging [33], RJ, which upregulates HIF-1α playing pivotal roles in the keratinocyte proliferation and EBM maintenance, is beneficial for anti-aging, as an ingredient of skin care products.