RIPK4 suppresses the TGF‐β1 signaling pathway in HaCaT cells

Abstract

Receptor‐interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor‐β 1 (TGF‐β1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF‐β signaling during epidermal homeostasis‐related events and suppression of RIPK4 expression by TGF‐β1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor‐κB (NF‐κB), protein kinase C (PKC), wingless‐type MMTV integration site family (Wnt), and (mitogen‐activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad‐mediated TGF‐β1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad‐mediated TGF‐β1 signaling events through suppression of TGF‐β1‐induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3‐Smad4 interaction, nuclear localization, and TGF‐β1‐induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound‐scratch assay demonstrated that RIPK4 suppressed TGF‐β1‐mediated wound healing through blocking TGF‐β1‐induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF‐β1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF‐β1 signaling.

A novel homozygous RIPK4 variant in a family with severe Bartsocas‐Papas syndrome

“…As previously reported, RIPK4, activates the NF‐κB and Wnt/β‐catenin signaling pathways, while suppressing the TGF‐β1/Smad signaling pathway (Dinçer et al, 2020; Huang et al, 2013; Meylan et al, 2002). Therefore, we performed the NF‐κB, Wnt/β‐catenin, and TGF‐β1/Smad‐regulated luciferase reporter assays to analyze the impact of p.Asp161His (D161H) substitution on RIPK4 kinase activity in those pathways.…”

“…Previously, it was demonstrated that RIPK4, but not kinase inactive variants, directly activates PKC, Wnt/β‐catenin, and NF‐κB signaling similar to that caused by ligands of these pathways (Huang et al, 2013; Kwa et al, 2014; Meylan et al, 2002). In addition to these findings, our recent study revealed for the first time that, instead of being an inducer, the RIPK4 acts as a negative regulator in TGF‐β1/Smad signaling pathway (Dinçer et al, 2020). Therefore, NF‐κB, Wnt/ β‐catenin, and TGF‐β1/Smad signaling pathways were selected in order to analyze the impact of identified p.Asp161His variant on RIPK4‐regulated signaling events.…”

“…RIPK4 communicates with different signaling pathways through its kinase activity, including PKC (Kwa et al, 2014), Wnt (Huang et al, 2013), NF‐κB (Meylan et al, 2002), Ras/MAP kinase (Lee et al, 2017), and TGF‐β1 (Dinçer et al, 2020); all of which play crucial roles for early development of many organs and tissues, including ectoderm (Lopez‐Pajares et al, 2013). Previously, it was demonstrated that RIPK4, but not kinase inactive variants, directly activates PKC, Wnt/β‐catenin, and NF‐κB signaling similar to that caused by ligands of these pathways (Huang et al, 2013; Kwa et al, 2014; Meylan et al, 2002).…”

“…The open reading frame of RIPK4 variants was amplified from pRC3‐VSV full‐length RIPK4 plasmid (Meylan et al, 2002) by using Phusion™ High‐Fidelity DNA Polymerase (Thermo Fisher Scientific) and cloned into the p3xFlagCMV/DEST mammalian expression vector infusion with FLAG® epitope by using Gateway®BP/LR Cloning system (Invitrogen). Cloning of wild type (NM_020639.2) and p.Lys51Arg RIPK4 into the p3xFlagCMV/DEST were performed as previously described by Dinçer et al, 2020. All the clones were verified by sequencing using the Genetic Analyzer (Applied Biosystems, ABI 3130).…”

Therapeutic candidates for keloid scars identified by qualitative review of scratch assay research for wound healing

“…•EGF [11,12] •Pep19-2.5 [13] •Hepatocyte Growth Factor (HGF) [14] •micro RNA 21 [15] •ERBB2 [16] •Keratinocyte Growth Factor (KGF) [17] •AES16-2M (ERK activating peptide) [18] •GFP-Smad2 [19] •Lipofectamine and KGF-mRNA [17] •SIS3 (Smad3 phosphorylation specific inhibitor) [19] •Liraglutide, a Glucagon-like peptide-1 analogue (concentration dependent) [20] •Thrombin [21] •AHR siRNA [22] •EGF receptor inhibitor (EGFRi) [19] •JNK inhibitor (JNKi) [19] •MEK1 inhibitor (MEKi) [19] •Enhanced green fluorescent protein (eGFP) with Lipofectamine [17] •LY 294002 (PI3K inhibitor) [20] •IDR-1018, a synthetic innate defense regulator peptide, in normoxia [23] •EGFR antagonist (AG1478) [21] •ERK1/2 antagonist (UO126) [21] •AHR antagonist, CH223191 [22] •RIPK4 via TGFβ [24] •ERBB3 [16] •ERK inhibitor (U0126) [18] •IDR-1018 in hypoxic condition [23] •ALK5 (TGF-ß receptor I) inhibitor (TGFRi) [19] •Cytochalasin D [22] •PXR siRNA [22] •PXR antagonist, SPA70 [22] •JNK inhibitor (SP600125 and indirubin) [22] Natural Products…”