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Page 6 of 10 Philips et al. Plast Aesthet Res 2022;9:4 https://dx.doi.org/10.20517/2347-9264.2021.83

EXTRACELLULAR MATRIX REMODELING
Oxidative damage and inflammation are associated with increased ECM remodeling, promoting skin
[63]
wrinkling and cancer progression . There is a coordinated regulation between inflammatory mediators
and the ECM proteins [63,64] . The primary structural ECM proteins are collagen and elastin, and the primary
ECM remodeling or degrading enzymes are matrix metalloproteinases (MMP) and elastase. A loss of
collagen and an increase in MMPs/elastases is associated with skin aging and cancer. Intrinsic aging is
associated with loss of elastin, whereas photoaging is associated with solar elastosis [65,66] . The action of MMPs
is based on substrate specificity or the elements in its promoters. Therefore, different MMPs can be found.
Based on substrate specificity, MPPs can be classified as: interstitial collagenases that cleave the fibrillar
collagens (predominantly MMP-1), the gelatinases (MMP-2 and MMP-9), and stromelysins (MMP-3 and
MMP-10) that cleave primarily the basement membrane, the membrane-type MMPs that cleave pro-MMPs,
[65]
and the other MMPs such as metalloelastase (MMP-12) that cleaves the basement membrane and elastin .
MMPs are alternatively also classified according to their regulatory promoter elements: group I that contain
TATA box and activator protein-1 (AP-1 site); group II, which bears no AP-1 site; and group III, which
display no TATA box or AP-1 sites . The transcription factor AP-1 is stimulated by the MAPK pathway,
[66]
which is activated by cellular inflammation and angiogenesis . MMP activity is inhibited by the tissue
[44]
inhibitors of matrix metalloproteinases (TIMP). The four TIMPs (TIMP-1, -2, -3, -4) bind to all of the
MMPs, though TIMP-1 has a preference for MMP-1 and TIMP-2 to MMP-2 . The TIMP-1 and TIMP-3
[67]
[67]
are inducible, TIMP-2 is constitutive, and TIMP-4 exhibits tissue specificity . ECM remodeling is
associated with increased expression of MMPs and elastase, and reduced expression of TIMPs and
collagen .
[67]
VD improves ECM proteins regulation in fibroblasts and melanoma cells [34,35] [Figure 2]. VD promotes the
expression of collagen, whereas it inhibits the expression of elastin [68,69] . VD stimulates the expression of
collagen by transcriptional mechanism, in non-irradiated and UVA-irradiated fibroblasts, though not in
UVB-irradiated fibroblasts . In UVA-irradiated fibroblasts, VD also stimulates heat shock protein-47
[35]
(HSP-47), a chaperone involved in the formation of collagen fibers, but again not in non-irradiated and
UVB-irradiated fibroblasts . VD also inhibits elastin promoter activity in non-irradiated and UV-
[35]
irradiated fibroblasts . It has also been described that VD inhibits elastase activity directly and its
[35]
expression in non-irradiated, and UVA-irradiated fibroblasts, though not in UVB-irradiated fibroblasts .
[35]
Elastase activity can be measured by incubating the enzyme with VD followed by the addition of its
substrate, whose degradation to a colored product can be followed by spectrophotometrically (Elastin
Products Co) . VD also inhibits MMP-1 and MMP-2 protein levels in melanoma cells .
[35]
[33]
The stated conclusions on the effects of VD on the extracellular matrix remodeling were determined
through in vitro experiments, in which non-irradiated, UVA-irradiated, or UVB-irradiated human dermal
fibroblasts, and melanoma cells were incubated for 24 h with different concentrations of VD, and
expression of different proteins was measured by RT-qPCR and/or ELISA. Protein levels of type I collagen,
elastin, MMP-1, and MMP-2 were measured in the media, and protein levels of HSP-47 were measured in
cells using ELISA. The RT-qPCR was used to measure mRNA levels of MMP-1 and MMP-2. Fibroblasts
were co-transfected with COL1α1 promoter-firefly luciferase or elastin promoter-firefly luciferase and TK
promoter-Renilla luciferase plasmids for 24 h, prior to the dosing with UV radiation and/or VD; and firefly
and renilla luciferase activities were measured sequentially to determine the normalized type I collagen or
elastin promoter activities. Melanoma cells were co-transfected with the MMP-1 promoter-
chloramphenicol acetyltransferase (CAT) plasmid and RSV2-β Galactosidase (β-GAL) prior to incubation
with or without VD, and the cells were examined for CAT expression and β-GAL activity to determine the
normalized MMP-1 promoter activity. Collectively, VD strengthens the ECM and is beneficial to the