Wrinkle formation and the loss of skin elasticity and plasticity occur over time and exposure to intracellular and extracellular stresses (Parrado et al., 2019). The extracellular matrix is particularly important in maintaining the integrity of the skin and possesses a complex structure composed of an interlocking mesh of fibrous proteins and proteoglycans. (Xue and Jackson, 2015). It is well-accepted that the activation of matrix metalloproteinases (MMPs) is responsible for promoting the structural alteration and degradation of fibrous proteins in the extracellular matrix as the skin undergoes aging (Freitas-Rodriguez et al., 2017).

MMPs are members of zinc-dependent endopeptidases and display proteolytic activity against a broad range of substrates (de Almeida et al., 2022). There are at least 23 paralogs of MMPs in humans, and they can be divided into at least six superfamilies: (1) collagenases, (2) gelatinases, (3) stromelysins, (4) metrylysins, (5) membrane-type MMPs, and (6) other MMPs (Klein and Bischoff, 2011). The typical structure of MMPs consists of an N-terminal zymogenic propeptide domain (~80 amino acids), a metal-dependent catalytic domain (~170 amino acids), a linker region (~15 – 65 amino acids), and a C-terminal hemopexin-like domain (~200 amino acids) (Fanjul-Fernandez et al. 2010). All MMPs are produced as proenzymes and require proteolytic cleavage to promote the release of the propeptide domain, e.g., zymogen activation (Quintero-Fabian et al., 2019). Significant efforts have been made to identify novel MMP inhibitors as drug candidates and cosmetic ingredients because the activation of MMPs is closely associated with the progression of skin aging and many chronic diseases (Cabral-Pacheco et al., 2020).

α-Ketoglutarate, a core metabolite in the tricarboxylic acid cycle (TCA), is an indispensable element required for the formation of adenosine triphosphate (ATP) in mitochondria (Wu et al., 2016). The production of α-KG occurs from the decarboxylation of isocitrate by isocitrate dehydrogenase or from the oxidative deamination of glutamate by glutamate dehydrogenase (Naeini et al., 2023). In addition to its role as an energy donor, α-ketoglutarate is also a key source of glutamate and glutamine for stimulating protein synthesis, scavenging nitrogen to prevent nitrogen overload, controlling gene expression by acting as a cofactor for histone and DNA demethylases and regulating the activity of prolyl hydroxylases (PHDs) (Bayliak and Lushchak, 2021). In the present study, we observed that the treatment of human keratinocyte HaCaT cells with dimethyl α-ketoglutarate (DMK), an alkylated derivative of α-ketoglutarate, increased the amount of intracellular α-ketoglutarate and 4-hydroxylproline and inhibited the expression of selected collagenases and elastases. These observations led us to conduct in-depth biochemical studies underlying how DMK protects against the skin wrinkle formation.

Cell culture, chemicals, antibodies, and plasmids

Dulbecco’s modified Eagle media (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (DPBS), and penicillin/streptomycin (Pen/Strep) were purchased from GenDEPOT (Austin, TX, USA). HaCaT cells were purchased from the Cell Lines Service GmbH (Dr.-Eckener-Str, Eppelheim, Germany). α-Ketoglutarate was purchased from Merck-Millipore (St. Louis, MO, USA). Dimethyl α-ketoglutarate was purchased from Bioscience (Cambridge, UK). HaCaT cells were cultured in 1× DMEM containing 10% FBS and 1% Pen/Strep at 37°C in a 5% CO₂ incubator. Antibodies against MMP-1, MMP-9, and MMP-12 were purchased from Proteintech (Rosemont, IL, USA). Antibodies against c-FOS, c-JUN, ERK1/2, and phospho-specific ERK1/2 at Thr202/204 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against actin and MMP-10 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The 4-hydroxyproline antibody was purchased from Biomatik (Wilmington, DE, USA).

Measurement of intracellular α-ketoglutarate

HaCaT cells were seeded at a density of 1×10⁶ cells in a 60 mm culture plate, and the amount of intracellular α-ketoglutarate was measured using an α-ketoglutarate assay kit (Cellbiolabs, San Diego, CA, USA), according to the manufacturer’s protocol.

MTT assay

HaCaT Cells were seeded at a density of 1×10⁴ cells in 96-well culture plates. After treatment, cells were washed with 1× DPBS and mixed with 10 μL MTT solution (5 mg/mL). After 2 h, 100 μL of DMSO was added, and the amount of dissolved formazan crystals was measured at a wavelength of 470 nm using a spectrophotometer.

RNA extraction and real-time reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted using the Hybrid-R RNA Extraction Kit (GeneAll, Seoul, Korea). cDNA was synthesized from total RNA (1 μg) using AmfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT). Real-time RT-PCR analysis was performed using SYBR mix (ELPIS Biotech, Daejeon, Korea) on a CFX384 real-time system, according to the manufacturer’s instructions (BioRad, Hercules, CA, USA). The mRNA level of individual genes was normalized by that of GAPDH. PCR primer sequences against individual genes are listed in Table 1.

Table 1 Sequences of the real-time RT-PCR primers

	Accession no.	Gene	Primer sequence
Human	NM_001289726.1	GAPDH	Forward: 5’-GGAGAGTGTTTCCTCGTCCC -3’ Reverse: 5’-ACTGTGCCGTTGAATTTGCC -3’
	NM_000917.4	Proyl-4-Hydroxylase	Forward: 5’ -CCGAGCTACAGTACATGACCC-3 Reverse: 5’-TGGCTCATCTTTCCGTGCAA-3
	NM_000088.4	COL1A1	Forward: 5’-TGCTCGTGGAAATGATGGTG-3 Reverse: 5’-CCTCGCTTTCCTTCCTCTCC-3
	NM_002421.4	MMP-1	Forward: 5’-TGTGGTGTCTCACAGCTTCC-3’ Reverse: 5’-TTGTCCCGATGATCTCCCCT-3’
	NM_004994.3	MMP-9	Forward: 5’-ACGATGACGAGTTGTGGTCC-3’ Reverse: 5’-TCGCTGGTACAGGTCGAGTA-3’
	NM_002425.3	MMP-10	Forward: 5’-AGTTAACAGCAGGGACACCG-3’ Reverse: 5’-GTCTAGGGAAGCCTTGCTCC-3’
	NM_002423.5	MMP-7	Forward: 5’-GGAGTGCCAGATGTTGC-3’ Reverse: 5’-ATCTCCTCCGAGACCTG-3’
	NM_002426.6	MMP-12	Forward: 5’-TTTGGTGGTTTTTGCCCGTG-3’ Reverse: 5’-ATGTCATCAGCAGAGAGGCG-3’
	NM_005252.4	c-Fos	Forward: 5’-CCTGCCTCTCCTCAATGACC-3’ Reverse: 5’-TCGGGGTAGGTGAAGACGAA-3’
	NM_002228.4	c-Jun	Forward: 5’-TATGACGATGCCCTCAACGC-3’ Reverse: 5’-CTGGATTATCAGGCGCTCCA-3’
	NM_000575.5	IL-1α	Forward: 5’-AGTAGCAACCAACGGGAAG-3’ Reverse: 5’-GCCGTGAGTTTCCCAGA-3’
	NM_000576.3	IL-1β	Forward: 5’-CTGAGCTCGCCAGTGAAAT-3’ Reverse: 5’-TCGTGCACATAAGCCTCG-3’
	XM_054341820.1	IL-1R	Forward: 5’- GGAGACGGAGGACTTGTGTG-3’ Reverse: 5’-ACTGGCCGGTGACATTACAG-3’

Immunochemistry

HaCaT cells were seeded at a density of 3×10⁵ cells in 24-well culture plates. After treatment, the cells were washed with 1× PBS and fixed with 4% paraformaldehyde. Cells were permeabilized with 0.1% Triton X-100. After washing three times with 1× PBS, cells were blocked with 5% bovine serum albumin (BSA) to remove non-specific binding and incubated overnight at 4°C with primary antibodies. After washing three times with 1× PBS, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated second antibody (GenDepot) for 1 h at 4°C. After washing twice, the cells were reacted with 4,6-diamidino-2-phenylindole (DAPI) for one min and mounted. Cells were visualized by confocal microscopy.

Measurement of human collagen

HaCaT cells were seeded at a density of 1×10⁶ cells in 60 mm culture plates, and the amount of human collagen 1α1 was measured using a FastScan™ COL1A1 ELISA Kit (Cell Signaling Technology) according to the manufacturer’s protocol.

Measurement of human collagenase-1

HaCaT cells were seeded at a density of 1×10⁶ cells in 60 mm culture plates, and the amount of collagenase-1 produced by HaCaT cells in the supernatant was measured using a Human Collagenase-1 ELISA Kit (MyBioSource, San Diego, CA, USA) according to the manufacturer’s protocol.

Western blot analysis

HaCaT cells were seeded at a density of 2×10⁶ cells in a 100 mm culture dish. After treatment, cell pellets were collected and resuspended in 1× RIPA lysis buffer [50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na3VO₄, 1 mM dithiothreitol (DTT), 1 mM phenylmethyl sulfonyl fluoride (PMSF)], and incubated on ice for 1 h. Cell lysates were collected by centrifugation at 13,000 rpm for 15 min, and protein concentration was measured using Pierce™ BCA Protein Assay Kits (Thermo Fisher Scientific, MA, USA). Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene membranes (Merck-Millipore Korea, Daejeon, Korea). The membranes were incubated in blocking buffer (5% skim milk in 1× PBS with 0.1% Tween-20, 1× PBST) for 1 h and hybridized with appropriated primary antibodies in 1× PBS overnight at 4°C. After washing three times with 1× PBST for 30 min, the membranes were hybridized with horseradish peroxidase-conjugated secondary antibodies (GenDEPOT) for 1 h at 4°C and washed three times with 1× PBST for 30 min. The membranes were visualized using an enhanced chemiluminescence detection system.

Measurement of human elastase

HaCaT cells were seeded at a density of 2×10⁷ cells in 60 mm culture plates, and the amount of human elastase was measured using a Human Neutrophil Elastase SimpleStep ELISA Kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol.

Generation of stable cells by lentiviral transduction

The pGreenFire-AP-1-GFP-luciferase lentiviral expression vector was purchased from Systems Biosciences (Palo Alto, CA, USA), and the lentiviral helper plasmids (pMD2.G and psPAX2) were acquired from Addgene (Cambridge, MA, USA). The generation of pGreenFire-AP1-GFP-luciferase plasmid is depicted in Fig. 4A. To perform lentiviral transduction in HaCaT cells, 293T cells were transfected with 1 μg of pGreenFire- AP-1-GFP-luciferase plasmid together with 1 μg of lentiviral helper plasmids (psPAX2 and pMD2.G). At 72 h post-transfection, the viral supernatant was collected, filtered, and transduced into HaCaT cells in the presence of 5 µg/mL polybrene (Merck-Millipore Korea). Transduced HaCaT cells were selected with puromycin (Invivogen, San Diego, CA, USA) at a concentration of 3 μg/mL for 48 h.

Figure 4. DMK inhibits the production of elastase and downregulates the expression of MMP-12 in HaCaT Cells. (A) Diagram depicting the role of MMP-7 and MMP-12 in the degradation of elastin. (B) HaCaT cells (2×10⁷ cells) were exposed to DMK at various times, and the production of elastase was measured by the Human Neutrophil Elastase Simple-Step ELISA Assay Kit (n=3). (C) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times and the mRNA levels of MMP-7 and MMP-12 were measured by real-time RT-PCR (n=4). (D) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the protein expression of MMP-12 was measured by Western blot analysis. **p<0.01 and ***p<0.001 vs Control (DMSO).

Firefly luciferase assay

pGreenFire-AP-1-GFP-luciferase cells were seeded at a density of 1×10⁵ cells in 24-well plates. After treatment, pGreenFire-AP1-GFP-luciferase cells were washed with 1× PBS and lysed with luciferase lysis buffer [0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, and 2 mM EDTA] for 1 h. The resulting firefly luciferase activity was monitored using the GLOMAX Multi-system (Promega, Madison, WI, USA). Luciferase activity was normalized to the protein concentration of the lysates.

Gene silencing of interleukin-1 receptor (IL-1R) with siRNA

IL-1R mRNA in HaCaT cells was knocked down by transfection with 10 μg IL-1R siRNA (Origene, Rockville, MD, USA). The IL-1R siRNA sequences, consisting of 21nt siRNA duplexes, are listed in Table 2.

Table 2 siRNA sequence used for transfection

Gene	Primer Sequence (5’→3’)
siIL-1R	Forward: GAG GAU UCA GGA CAU UAC U Reverse: AGU AAU GUC CUG AAU CCU C

Statistical analysis

Statistical analyses were conducted by the Student’s t-test using the Microsoft Excel program (Microsoft, Redmond, WA, USA). Asterisks in the figures indicate statistical significance with *p<0.05, **p<0.01, and ***p<0.001.

Treatment with dimethyl α-ketoglutarate (DMK) increases the amount of intracellular α-ketoglutarate in HaCaT cells

We exposed HaCaT cells to α-ketoglutarate and DMK (Fig. 1A) and compared the amount of intracellular α-ketoglutarate. We speculated that DMK would permeate the cellular membrane better than α-ketoglutarate due to an increase in hydrophobicity and hydrolyzed inside the cells. As expected, we observed that treatment with DMK increased the amount of intracellular α-ketoglutarate significantly more than that of α-ketoglutarate (Fig. 1B) without affecting the viability of HaCaT cells (Fig. 1C). These results suggest that DMK treatment could be used as a strategy to increase the amount of intracellular α-ketoglutarate.

Figure 1. DMK increases the level of 4-hydroxyproline and promotes the synthesis of collagen in HaCaT cells. (A) Diagram depicting the conversion of proline to hydroxyproline by prolyl 4-hydroxylase (P4H). (B) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the mRNA level of P4H was measured by real-time RT-PCR (n=4). (C) HaCaT cells (1×10⁵ cells) were exposed to DMK for 24 h, and the level of 4-hydroxyproline was measured by an immunocytochemical assay. (D) HaCaT cells (1×10⁶ cells) were exposed to DMK at various times, and the production of collagen type 1α1 (COL1A1) was measured by the collagen type 1α1 ELISA assay (n=3). (E) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the mRNA level of COL1A1 was measured by real-time RT-PCR (n=4). *p<0.05, **p<0.01, and ***p<0.001 vs Control (DMSO).

Treatment with DMK increases 4-hydroxyproline and the synthesis of collagen in HaCaT cells

Collagen is composed of a repeating tripeptide (Glycine-X-Y), in which X and Y are usually proline or 4-hydroxyproline residues (Ramshaw et al., 1998). 4-Hydroxyproline plays an essential role in the folding of collagen, and prolyl 4-hydroxylase (P4H) catalyzes the conversion of proline to 4-hydroxyproline using α-ketoglutarate and ferrous iron (Fe²⁺) as substrates (Fig. 2A). Because treatment with DMK increased the intracellular amount of α-ketoglutarate (Fig. 1B), we assumed that this would increase the formation of 4-hydroxyproline and, possibly, the stabilization of collagen. Consistent with this speculation, we observed that treatment with DMK caused the transcriptional activation of P4H (Fig. 2B) and increased the amount of cellular 4-hydroxyproline (Fig. 2C) in HaCaT cells. Treatment with DMK also increased the amount of collagen type 1α1 (COL1A1) (Fig. 2D) via transcriptional activation (Fig. 2E). Together, these data illustrate that treatment with DMK increased the amount of 4-hydroxyproline and stimulated the synthesis of collagen in HaCaT cells.

Figure 2. DMK increases the production of α-ketoglutarate in HaCaT cells without affecting the viability of HaCaT cells. (A) Chemical structures of α-ketoglutarate and dimethyl α-ketoglutarate (DMK). (B) HaCaT cells (1×10⁶ cells) were exposed to α-ketoglutarate and DMK at various times, and the amount of intracellular α-ketoglutarate was measured (n=3). (C) HaCaT cells (1×10⁴ cells) were exposed to DMK at various concentrations, and the viability was measured by the MTT assay (n=3). *p<0.05 and **p<0.01 vs Control (DMSO).

Treatment of DMK inhibits MMP-1, MMP-9, and MMP-10 in HaCaT cells

The MMPs have evolved into different groups by remodeling some conserved domains or by incorporating other domains absent in MMPs. They are subdivided according to substrate specificity, sequence homology, and domain organization and include collagenases, stromelysins, and gelatinases (Geervliet and Bansal, 2020). Collagen is cleaved by collagenase (MMP-1, MMP-8, MMP-13, and MMP-18) and stromelysins (MMP-3 and MMP-10), and the cleaved collagens can be further fragmented into smaller peptides by gelatinases (MMP-2 and MMP-9) (Fig. 3A) (Bohn et al., 2016). Collagenase and stromelysins are composed of a peptide, catalytic domain, linker region, and hemopexin-like domain, whereas gelatinases have three additional fibronectin type II domains within the catalytic domain (Uria and Werb, 1998). In the present study, we observed that treatment with DMK suppressed the production of collagenase in HaCaT cells (Fig. 3B). Treatment with DMK inhibited transcription (Fig. 3C) and the expression (Fig. 3D) of selected collagenases (MMP-1, MMP-9, and MMP-10). Together, these results illustrate that treatment of DMK inhibited the production of collagenase and the expression of selected MMPs (MMP-1, MMP-9, and MMP-10) in HaCaT cells.

Figure 3. DMK inhibits the production of collagenase and downregulates the expression of MMP-1, MMP-9, and MMP-10 in HaCaT Cells. (A) Diagram depicting the types and roles of various matrix metalloproteinases in the degradation of collagen. (B) HaCaT cells (5×10⁶ cells) were exposed to DMK at various times, and the production of collagenase was measured by the human collagenase-1 ELISA assay (n=3). (C) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the mRNA levels of MMP-1, MMP-9, and MM-10 were measured by real-time RT-PCR analysis (n=4). (D) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the protein expression of MMP-1, MMP-9, and MMP-10 was measured by Western blot analysis. **p<0.01 and ***p<0.001 vs Control (DMSO).

Treatment of DMK inhibits MMP-12 in HaCaT cells

Elastin is an essential component of extracellular matrix proteins and exists as a core protein of elastic fibers (Heinz, 2021). Elastin is essential for maintaining the integrity and plasticity of the skin (Heinz, 2020), and the enzymatic degradation of elastin occurs as a result of the upregulation of elastases such as MMP-7 and MMP-12 (Fig. 4A). We observed that treatment with DMK significantly inhibited the production of elastase in HaCaT cells (Fig. 4B), and this event was associated with the transcriptional inhibition of MMP-12 but not MMP-7 (Fig. 4C). Consistent with this, treatment with DMK suppressed the protein expression of MMP-12 in HaCaT cells (Fig. 4D). Together, our results illustrate that treatment with DMK inhibited MMP-12 and suggest that it might contribute to a decrease in the production of elastase in HaCaT cells.

Treatment of DMK inhibits AP-1 by suppressing the expression of c-Jun in HaCaT cells

Since the promoter of most MMP genes harbors an activator protein-1 (AP-1) site in the proximal promoter located close to a typical TATA box (Yan and Boyd, 2007), we attempted to examine whether treatment with DMK could inhibit AP-1. To this end, we established HaCaT-AP-1-GFP-luciferase cells by lentiviral transduction, followed by puromycin selection (Fig. 5A), and exposed HaCaT-AP-1-GFP-luciferase cells to varying concentrations of DMK. As a result, we found that treatment with DMK significantly inhibited AP-1-dependent luciferase activity (Fig. 5B). Treatment with DMK also suppressed the expression of c-JUN but not that of c-FOS (Fig. 5C), and it was closely associated with the transcriptional inhibition of c-Jun (Fig. 5D). These results indicate that transcriptional inhibition of c-Jun was responsible for the inhibition of AP-1 by DMK in HaCaT cells.

Figure 5. The inhibition of c-Jun and selected MMPs by DMK is attributable to the attenuation of the IL1α/β-IL1R-ERK1/2-AP1 axis in HaCaT cells. (A) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the mRNA levels of IL-1α and IL-1β were measured by real-time RT-PCR analysis (n=4). (B) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the mRNA levels of IL-1R were measured by real-time RT-PCR analysis (n=4). (C) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the level of ERK1/2 phosphorylation was measured by Western blot analysis. (D) The mRNA levels of c-Jun were measured in HaCaT cells by real-time RT-PCR after IL-1R was knocked down (n=4). (E) The mRNA levels of MMP-1, MMP-9, MMP-10, and MMP-12 were measured in HaCaT cells by real-time RT-PCR after IL-1R was knocked down (n=4). *p<0.05, **p<0.01, and ***p<0.001 vs Control (DMSO).

The IL-1 receptor is responsible for mediating the inhibition of AP-1 by DMK in HaCaT cells

Interleukin-1α/β (IL-1α and IL-1β) are pro-inflammatory cytokines that regulate cutaneous inflammation and mediate various physiological responses (Di Paolo and Shayakhmetov, 2016). IL-1α and IL-1β are alarm cytokines (known as alarmins) that act through the IL-1 receptor (IL-1R), thereby initiating and amplifying local inflammatory responses (Briukhovetska et al., 2021). To examine whether interleukins and their receptor were associated with the inhibition of AP-1 by DMK, we measured IL-1α and IL-1β mRNA levels in HaCaT cells after treatment with DMK. Treatment with DMK decreased IL-1α and IL-1β mRNA levels (Fig. 6A) and also downregulated the level of IL-1R mRNA (Fig. 6B) and its downstream target, ERK1/2 phosphorylation at Thr202/204 (Fig. 6C). Consistent with these observations, knocking down IL-1R in HaCaT cells downregulated the mRNA level of c-Jun (Fig. 6D) and that of MMP-1, MMP-9, MMP-10, and MMP-12 (Fig. 6E) Together, these results imply that the inhibition of IL-1α/β and the IL-1R by DMK might be responsible for the inhibition of AP-1 and selected MMPs in HaCaT cells.

Figure 6. DMK inhibits AP-1 transactivation in HaCaT cells. (A) Diagram demonstrating the generation of pGreenFire-HaCaT-AP-1-GFP-luciferase cells. (B) Established HaCaT-AP-1-GFP-luciferase cells (1×10⁶ cells) were exposed to DMK for 24 h at various concentrations, and AP-1-dependent luciferase activity was measured by the luciferase assay (n=3). (C) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the protein expression of c-JUN and c-FOS was measured by Western blot analysis. (D) HaCaT cells (2×10⁶ cells) were exposed to DMK at various times, and the mRNA levels of c-Fos and c-Jun were measured by real-time RT-PCR (n=4). **p<0.01 and ***p<0.001 vs Control (DMSO).

Collagen has evolved to provide tensile strength in connective tissues (Vasta and Raines, 2018). Dietary ascorbate (vitamin C) is essential for maintaining the folding of collagen and the activity of prolyl 4-hydroxylase by preventing Fe²⁺ oxidation (Fig. 2A). The importance of this process is clinically manifested in scurvy, a disease characterized by the deformation of the collagen structure due to a lack of ascorbate (Gandhi et al., 2023). Considering the importance of prolyl 4-hydroxylase in maintaining the structure of collagen, we assumed that supplementation with intracellular α-ketoglutarate, a substrate of prolyl 4-hydroxylase, would increase the amount of intracellular 4-hydroxyproline. Treatment with DMK not only increased the amount of 4-hydroxyproline in HaCaT cells (Fig. 2C) but also promoted the synthesis of collagen (Fig. 2D) via transcriptional activation (Fig. 2E).

Treatment with DMK also suppressed the production of collagenase (Fig. 3B) and the expression of selected MMPs (MMP-1, MMP-9, and MMP-10) (Fig. 3C, 3D) in HaCaT cells, while it failed to inhibit the other MMPs listed in Fig. 3A (data not shown). Likewise, treatment with DMK suppressed the production of elastase in HaCaT cells (Fig. 4B) and the expression of MMP-12 but not that of MMP-7 (Fig. 4C, 4D). Presently, the molecular mechanisms responsible for the selective inhibition of MMPs by DMK are unclear, but we speculate that the existence of differential binding sites of transcription factors in the promoter might be responsible, at least in part, for the downregulation of selected MMPs by DMK. For example, we observed that treatment with DMK inhibited the expression of MMP-1 and MMP-9 (Fig. 3C, 3D) but not that of MMP-2 (data not shown), and this finding correlates well with the existence of an AP-1 binding site in the promoter: MMP-1 and MMP-9, but MMP-2, harbor an AP-1 binding site in the promoter (Fanjul-Fernandez et al., 2010). However, the possibility that transcriptional factors other than AP-1 might have participated in the selective regulation of MMPs by DMK can’t be excluded.

Many previous studies have demonstrated the beneficial effects of α-ketoglutarate. α-Ketoglutarate is an antioxidant in vitro and in vivo (He et al., 2018). The administration of α-ketoglutarate extended the lifespan and decreased morbidity in mice, and dietary supplementation with α-ketoglutarate increased muscle strength, exhibited cardioprotective effects, and protected against osteoporosis in humans (Asadi Shahmirzadi et al., 2020). Because DMK inhibited the expression of selected MMPs (Fig. 3, 4) and AP-1 (Fig. 5), it is conceivable that α-ketoglutarate would exert anti-tumor effects, and previous studies support this assumption. α-Ketoglutarate protected against tumor progression (Abla et al., 2020) and metastasis and sensitized cells to the antineoplastic effects of oxidative phosphorylation inhibitors (Sica et al., 2019). Together, we demonstrated that supplementing intracellular α-ketoglutarate with a cell-permeable derivative of α-ketoglutarate (DMK) could serve as a strategy to stabilize the collagen structure, promote collagen synthesis, and inhibit MMPs in HaCaT cells. These findings suggest that DMK might be useful as a feasible anti-wrinkle candidate.

This research was supported by the National Research Industrial Cluster Competitiveness Reinforcement project funded by the Korea Industrial Complex Corporation (KICOX, IRIC2210).

We declare no conflicts of interest in the present study.