Selective Hyaluronan-CD44 Signaling Promotes miRNA-21 Expression and Interacts with Vitamin D Function during Cutaneous Squamous Cell Carcinomas Progression Following UV Irradiation

Abstract

Hyaluronan (HA), the major extracellular matrix component, is often anchored to CD44, a family of structurally/functionally important cell surface receptors. Recent results indicate that UV irradiation (UVR)-induced cutaneous squamous cell carcinomas (SCC) overexpress a variety of CD44 variant isoforms (CD44v), with different CD44v isoforms appear to confer malignant SCC properties. UVR also stimulates HA degradation in epidermal keratinocytes. Both large HA polymers and their UVR-induced catabolic products (small HA) selectively activate CD44-mediated cellular signaling in normal keratinocytes and SCC cells, with all of the downstream processes being mediated by RhoGTPases (e.g., Rac1 and Rho). Importantly, we found that the hormonally active form of vitamin D 1,25(OH)2D3 not only prevents the UVR-induced small HA activation of abnormal keratinocyte behavior and SCC progression, but also enhances large HA stimulation of normal keratinocyte activities and epidermal function(s). The aim of this hypothesis and theory article is to question whether matrix HA and its UVR-induced catabolic products (e.g., large and small HA) can selectively activate CD44-mediated cellular signaling such as GTPase (Rac and RhA) activation. We suggested that large HA-CD44 interaction promotes Rac-signaling and normal keratinocyte differentiation (lipid synthesis), DNA repair, and keratinocyte survival function. Conversely, small HA-CD44 interaction stimulates RhoA activation, NFκB/Stat-3 signaling, and miR-21 production, resulting in inflammation and proliferation as well as SCC progression. We also question whether vitamin D treatment displays any effect on small HA-CD44v-mediated RhoA signaling, inflammation, and SCC progression, as well as large HA-CD44-mediated differentiation, DNA repair, keratinocyte survival, and normal keratinocyte function. In addition, we discussed that the topical application of signaling perturbation agents (e.g., Y27623, a ROK inhibitor) may be used to treat certain skin diseases displaying upregulation of keratinocyte proliferation such as psoriasis and actinic keratoses in order to correct the imbalance between Rac and RhoA signaling during various UV irradiation-induced skin diseases in patients. Finally, we proposed that matrix HA/CD44-signaling strategies and matrix HA (HAS vs. HAL or HAS → HAL)-based therapeutic approaches (together with vitamin D) may be used for the treatment of patients suffering a number of UV irradiation-induced skin diseases (e.g., inflammation, skin cancer, and chronic non-healing wounds).

Keywords: CD44; RhoGTPase; UVR; hyaluronan; miR21; skin cancer; vitamin D.

Figure 1

Detection of CD44 isoform expression in normal keratinocytes and SCC cell lines using anti-CD44-mediated immunoblotting techniques [SCC-4, SCC-12 cell lines, and HACAT (a transformed cell line) Epican is designated as a CD44v3-10 form; anti-CD44-mediated immunoblot was used to detect CD44 signal as indicated].

Figure 2

(A) Immunoperoxidase staining of CD44v3 and CD44v6 isoforms in normal and SCC tissues of human skin; and (B) immunoperoxidase staining of CD44v3 isoform in mouse skin following acute or chronic UVR exposure.

Figure 3

Measurement of HA-size distribution in untreated mouse epidermis vs. acute UVB-treated mouse epidermis. HA from untreated mouse epidermis vs. acute UVB-treated mouse epidermis was isolated and purified using gel filtration column chromatography, and then analyzed by an enzyme-linked binding protein assay, that uses microwells coated with a highly specific HA binding protein (HABP) to capture HA, and an enzyme-conjugated HABP using a commercially available enzyme-linked immunosorbent assay (ELISA)-type test kit. HA was also be analyzed by 0.5% agarose gel followed by Alcian blue and silver staining.

Figure 4

(A) Immunoblot analyses of large HA and PKN-dependent phosphorylation of p38MAPK and AP-1 proteins (e.g., c-Jun) [the PKN-dependent phosphorylation is proven by the effect of PKNγ-ACC cDNA dominant negative mutant], and (B) immunoblot analyses of large HA and vitamin D-induced c-Jun phosphorylation and differentiation marker (involucrin and transglutaminase) expression in cultured keratinocytes.

Figure 5

Immunoperoxidase staining of cytokine (IL-6) and chemokine (MCP-1) in mouse skin following acute or chronic UVR exposure.

Figure 6

(A) Illustration of 130-bp regions containing two predicted Stat-3 binding sites (site 1 and site 2) upstream of the miR-21 genes; (B) ChIP assay of SCC-12 cells treated with no HA (lane 1) or with small HA (lane 2) using anti-PKCε, anti-Stat-3, or IgG control. Co-immunoprecipitated DNA was amplified by PCR with primers specific for the miR-21 upstream promoter enhancer.

Figure 7

UVB-induced miR-21 production using RNase protection assay in cultured keratinocytes [treated with no UVB (lane 1) or with UVB (lane 2) or treated with ROK inhibitor (Y27632) plus UVB (lane 3) or treated with UVB and vitamin D (lane 4)], and in UVB-induced SCC tumor tissues (lane 5) or normal skin tissues (lane 6).

Figure 8

Detection of UVB-induced miR-21 expression in wild-type (VDR^+/+) and VDR-null (VDR^−/−) mouse skin using DIG-labeled miR-21 probe (LNA probe) (A–F) (and scrambled probe) (a & b) and in situ hybridization [incubated with an anti-DIG-AP (alkaline phosphatase)-FAB fragment plus NBT/BCIP].

Figure 9

Large HA-mediated upregulation of signaling regulators in keratinocytes. Murine keratinocytes isolated from epidermal sheets were pretreated with HA (50 μg/ml) (lane 2) in the presence or absence of p38MAPK inhibitor (SB203580) for 24 h followed by irradiation with UVB (708 J/m²), harvested and analyzed by immunoblotting using anti-p38MAPK (a) or anti-p63 antibody (b) or anti-VDR antibody (c) or anti-XPC antibody (d) respectively. Actin was probed by anti-actin antibody as a loading control. (Cells were treated with no HA (lane 1) or with large HA (lane 20) or with anti-CD44 plus large HA (lane 3) or with SB203580 plus large HA (lane 4) or with small HA (lane 5) [p63, a p53-like transcription factor; VDR, vitamin D receptor; XPC, DNA repair protein].

Figure 10

A proposed model for UVR-induced hyaluronan (HA) degradation and selective HA (small HA vs. large HA)-CD44 interaction with RhoA–ROK and Rac1-PKNγ in regulating normal and transformed keratinocyte signaling leading to healthy epidermal function and/or cutaneous squamous cell carcinomas (SCC) progression. Vitamin D enhances large HA-CD44-mediated differentiation, DNA r8epair, and keratinocyte survival/normal epidermal function, and it also inhibits small HA/CD44v-mediated RhoA–ROK signaling and SCC-specific tumor behaviors.