BackgroundKeratinocytes are the major cell type building human epidermis, which undergo coordinated processes of division and differentiation. They proliferate in the basal layer, then commit to differentiate and migrate towards the surface of the epidermis1. On the molecular level, basal keratinocytes highly express keratin 5 (KRT5) and keratin 14 (KRT14). This pattern is then switched to keratin 1 (KRT1) and keratin 10 (KRT10) as cells commit to differentiation2. During terminal differentiation, filaggrin (FLG), involucrin (IVL), and transglutaminase-1 (TGM1) are highly expressed and play essential role in the formation of the protective skin barrier3. Mechanistically, the KRT5-KRT14 intermediate filament (IF) pair is essential for the maintenance of the structural integrity of the basal layer, whereas the KRT1-KRT10 one supports the structure of the suprabasal epidermis4,5. Disturbed expression of those keratin genes is a hallmark of many skin diseases, particularly those involving altered proliferation and differentiation of keratinocytes6.We recently demonstrated that monocyte chemotactic protein-induced protein 3 (MCPIP3), also known as Regnase-3 modulates processes related to keratinocyte proliferation and differentiation7. MCPIP3 is encoded by the ZC3H12C gene and has RNase properties. It belongs to the MCPIP family of CCCH-type zinc finger proteins, acting as endonucleases and degrading specific mRNAs, thereby regulating gene expression post-transcriptionally8,9,10. The MCPIP family, especially MCPIP1, is central to immune regulation, inflammation resolution, antiviral defense, and tissue homeostasis11,12. While MCPIP1 is well-studied, the functions of MCPIP2–4 still require further investigation to fully elucidate their roles in health and disease.MCPIP1 and MCPIP3 proteins have been identified as regulators of epidermal biology13,14,15,16. Loss of MCPIP1 in mouse epidermis upregulates the levels of transcripts related to inflammation and keratinocyte differentiation, leading to the development of local and systemic inflammation13. Our recent studies utilizing a mouse model with keratinocyte-specific deletion of MCPIP3 indicated that the activity of MCPIP3 in keratinocytes is also essential for the maintenance of proper epidermal structure and function, but the mechanism is completely different7. In contrast to MCPIP1-specific deletion, MCPIP3 keratinocyte-specific knockout mice did not develop skin inflammation. However, their epidermis showed elevated proliferation rate. On the molecular level, increased expression level of key factors promoting nuclear division was observed in their skin. In vitro studies on human keratinocytes showed that silencing of MCPIP3 slightly increases their viability suggesting an impact of MCPIP3 on cell cycling/proliferation7.Unperturbed activity of MCPIP3 in keratinocytes is important for the proper control of their proliferation and differentiation program. When this process is unbalanced, it can lead to the development of skin diseases. The aim of the current study was to explore the mechanisms of MCPIP3-dependent effects on keratinocytes, with the focus on identification of its binding partners using immunoprecipitation-proteomics approach and investigating its role in cell cycle progression.Materials and methodsCell cultureHuman immortalized HaCaT keratinocytes17 were cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA). The cells were grown at 37 °C and 5% CO2 humidified atmosphere.TransfectionHaCaT cells were seeded at a density of 8 × 105 cells per well of a 6-well plate 24 h prior to transfection with 20 nM siGENOME Human ZC3H12C (#85463) SMARTpool, siGENOME Human KRT14 (#3861) SMARTpool or siGENOME Non-Targeting siRNA Pool #1 (Dharmacon) using JetPrime reagent (Polypus, London, UK) and collected after 72 h for RT-qPCR, western blot, flow cytometry or immunofluorescence analyses.Cell synchronizationHaCaT cells were seeded at 40% confluency and treated with 2 mM thymidyne (Sigma-Aldrich) for 24 h. The cells were then washed with PBS and cultured in complete DMEM for 12 h prior to addition of 2 mM thymidine for 24 h. The cells were then washed and harvested at specific intervals for flow cytometry or western blot analyses.ImmunoprecipitationThe doxycycline-dependent TetON system was used (pLIX vectors) to obtain HaCaT cells overexpressing 3xFLAG-MCPIP3, or empty control, as described previously18. In parallel, HaCaT cells overexpressing 3xFLAG-MCPIP1, or empty control, were used as an experimental control (data not shown). To induce expression of exogenous protein, cells were stimulated with doxycycline (BioShop, Burlington, Canada). For immunoprecipitation, cells grown at sub-confluency were lysed in IP lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris pH 7.5 with protease and phosphatase inhibitors). For mass spectrometric analysis, protein lysates were incubated with anti-FLAG M2 (Sigma-Aldrich) coated Protein G Dynabeads (Invitrogen, Darmstadt, Germany) for 2 h at 4 °C, followed by competitive elution with 1.5 mg/ml 3xFLAG peptide (Invitrogen). Eluates were snap-frozen and stored at −80 °C. For western blot, the beads were mixed with sample buffer and boiled for 5 min at 65 °C to elute proteins.Mass spectrometry analysisMass Spectrometry analysis was performed by the Core Facility for Proteomics at Medical University of Vienna. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE19 partner repository with the dataset identifier PXD067727.Immunoprecipitation eluates, prepared in triplicates, for 3xFLAG-MCPIP3 vs. experimental control and for 3xFLAG-MCPIP1 vs. experimental control (data not shown) were subjected to Mass Spectrometry analysis. The samples were reduced, alkylated and bound to the SP3 beads (GE Healthcare, Amersham, UK). Next, they were subjected to on-bead digestion with trypsin/LysC Mix (Promega, Walldorf, Germany) overnight at 37 °C in 50 mM ammonium bicarbonate, pH 8.5 (Sigma-Aldrich). After elution peptides were desalted using Pierce Peptide Desalting spin columns (Thermo Fisher Scientific, Waltham, MA, USA). The elutions were dried in a vacuum concentrator and reconstituted in 0.1% trifluoroacetic acid. LC-MS was performed on an Ultimate 3000 RSLC nano coupled directly to an Exploris 480 with FAIMSpro (all Thermo Fisher Scientific). MS scans were performed in the range from m/z 375–1650 at a resolution of 60,000 (at m/z = 200). MS/MS scans were performed choosing a resolution of 15,000; normalized collision energy of 29%; isolation width of 1.4 m/z and dynamic exclusion of 90s. Two different FAIMS voltages were applied (−40 V and − 60 V) with a cycle time of 1.5 s per voltage. The acquired raw MS data files were processed and analyzed using ProteomeDiscoverer (v2.4.0.305, Thermo Fisher Scientific). Precursor ion quantification was done using the Minora Feature Detector node. Only unique peptides were used for quantification, which was based on intensity. Normalization was done on total peptide amount and scaling mode on all average. Only peptides and proteins with FDR