EGF plays an important role in regulating skin cell homeostasis and proliferation of keratinocyte. It is also a potential regulator of inflammatory and proliferative responses [16]. Receptor tyrosine-protein kinase erbB-4 (ERBB4), a member of the EGF receptor family, is phosphorylated in HaCaT cells following EGF stimulation. ERBB4-knockout mice lack EGF-stimulated signaling, resulting in reduced cell proliferation and significantly reduced epidermal thickness [17]. Collagen is a constituent of the ECM and is located in fibrous tissues such as the skin, ligament, tendon, cornea, blood vessels, bone, and cartilage. Recent studies have shown that collagen promotes cell proliferation, differentiation, expression, and formation of tissues and organs [18].
Many studies have shown that MSCs promote cell proliferation, adhesion, wound healing, and reduce inflammation [19, 20]. Human umbilical MSCs promote human retinal progenitor cell adherence and differentiation toward retinal neurons [21]. Therefore, we investigated whether hEGF MSCs affect human keratinocyte HaCaT cell proliferation via collagen. In the present study, we first confirmed that hEGF MSCs promote keratinocyte proliferation and migration unlike MSCs. The results also revealed that cell viability rates were increased following treatment with 8% CM from hEGF MSCs (Fig. 3). Moreover, we examined the migration of keratinocytes treated with hEGF MSC-CM using the wound-healing assay. The results showed that wound-healing rates also increased (Fig. 4). These results suggest that hEGF MSCs promote cell proliferation and migration in keratinocytes. In general, when cells migrate to the wound, they reconstitute the actin cytoskeleton and secrete various proteases such as MMP-2 and MMP-9 to remodel the ECM [22]. Upregulation of MMP-1 degrades collagen, which is associated with skin conditions and adversely affects skin cell proliferation. Therefore, we examined the changes in the expression of MMP-1 (collagenase), an ECM protease, in hEGF MSC-CM-treated keratinocytes.
Inhibition of collagenase activity is crucial for preventing skin aging [23]. Furthermore, vitamin C has been reported to have an anti-aging effect on the skin through the inhibition of MMP-1, a well-known collagenase [24]. These studies suggest that inhibition of collagenase may be a key factor in preventing skin aging. MMP-1 breaks down type-I collagen by cleaving peptide bonds between Gly-Ile or Gly-Lys of triple helical collagen monomers [25]. To investigate the major factors that affect collagen, including MMP-1, we conducted zymography, western blotting, and qPCR assays. The results showed that the level of collagenase activity and expression level of MMP-1 mRNA and protein were decreased in hEGF MSC-CM-treated keratinocytes (Fig. 6). These results suggest that EGF represents a potentially effective factor in preventing skin aging.
Previous studies have shown that the MAPK pathway regulates MMP-1 expression. For example, cigarette smoke induces the upregulation of MMP-1 expression via the MAPK pathway [14]. Upregulation of MMP-1 is also regulated by the ERK pathway in melanoma cells [26]. Additionally, EGF mediates the expression of MMP-1. Suppression of the EGF ligand regulated the expression of MMP-1 in breast cancer cells [27]. Many studies have shown the involvement of the Akt pathway in cell proliferation and development [28]. Therefore, we performed western blotting to determine the protein expression levels of MAPK (Erk, JNK, and c-Fos) and Akt. The results revealed that MAPK expression levels (phosphorylated forms) were decreased and pAkt was increased (Fig. 7A). Moreover, the expression levels of the other transcription factors were also decreased (Fig. 7B).
2-DE is effective in separating proteins and protein subunits using isoelectric focusing in one dimension and molecular weight in the second dimension [29]. Therefore, we conducted 2-DE proteomic analysis and MALDI-TOF to identify the differentially expressed proteins following treatment of keratinocytes with Vector MSC-CM and hEGF MSC-CM. Approximately 60 protein spots were detected (Fig. 5). Among the differentially expressed proteins spots, carboxypeptidase, which was upregulated, is well-known to promote collagen synthesis [30], fibroblast proliferation [31], and differentiation into myofibroblast [32]. For example, the aortic carboxypeptidase-like protein, which is an ECM protein, is expressed in collagen-rich tissues and promotes embryonic development and dermal wound healing during mouse embryogenesis [33]. Therefore, we hypothesize that upregulation of carboxypeptidase affects HaCaT cell proliferation induced by hEGF MSCs.