The Nuclear Localization Signal of NF-κB p50 Enters the Cells via Syndecan-Mediated Endocytosis and Inhibits NF-κB Activity

It is well established that cationic peptides can enter cells following attachment to polyanionic membrane components. We report that the basic nuclear localization signal (NLS) of the NF-κB p50 subunit is internalized via lipid raft-dependent endocytosis mediated by heparan sulfate proteoglycans and exerts significant NF-κB inhibitory activities both in vitro and in vivo. In vitro uptake experiments revealed that the p50 NLS peptide (CYVQRKRQKLMP) enters the cytoplasm and accumulates in the nucleus at 37 °C. Depleting cellular ATP pools or decreasing temperature to 4 °C abolished peptide internalization, confirming the active, energy-dependent endocytic uptake. Co-incubation with heparan sulfate or replacing the peptide’s basic residues with glycines markedly reduced the intracellular entry of the p50 NLS, referring to the role of polyanionic cell-surface proteoglycans in internalization. Furthermore, treatment with methyl-β-cyclodextrin greatly inhibited the peptide’s membrane translocation. Overexpression of the isoforms of the syndecan family of transmembrane proteoglycans, especially syndecan-4, increased the cellular internalization of the NLS, suggesting syndecans’ involvement in the peptide’s cellular uptake. In vitro, p50 NLS reduced NF-κB activity in TNF-α-induced L929 fibroblasts and LPS-stimulated RAW 264.7 macrophages. TNF-α-induced ICAM-1 expression of HMEC-1 human endothelial cells could also be inhibited by the peptide. Fifteen minutes after its intraperitoneal injection, the peptide rapidly entered the cells of the pancreas, an organ with marked syndecan-4 expression. In an acute pancreatitis model, an inflammatory disorder triggered by the activation of stress-responsive transcription factors like NF-κB, administration of the p50 NLS peptide reduced the severity of pancreatic inflammation by blocking NF-κB transcription activity and ameliorating the examined laboratory and histological markers of pancreatitis.


Introduction
Peptide-mediated import of biomolecules has become a popular approach to modulating cellular functions (Heitz et al. 2009;Bohmova et al. 2018;Sanchez-Navarro 2021;Shoari et al. 2021;Yokoo et al. 2021;). Efficient intracellular delivery of bioactive compounds (peptides, oligonucleotides, etc.) with cell-penetrating peptides (CPPs)-small cationic peptides readily translocating through cell membranes and delivering attached cargoes intracellularlyopened up new possibilities for therapeutic interventions (Dougherty et al. 2019;Xie et al. 2020;Liu et al. 2021;Tarvirdipour et al. 2021). At the time of CPP discovery, endocytosis-due to lysosomal degradation of internalized agents-was viewed as an entry mechanism to be avoided for the intracellular delivery of bioactive agents. Thus, early studies claimed endocytosis-independent cellular internalization of CPPs (Derossi et al. 1994(Derossi et al. , 1996(Derossi et al. , 1998Oehlke et al. 1997;Vives et al. 1997;Letoha et al. 2003). However, the broadening knowledge of the newly explored lipid raft-and caveolae-mediated endocytic pathways helped to redefine the molecular mechanism of CPPs' efficient cellular entry and avoidance of lysosomal degradation (Nichols and Lippincott-Schwartz 2001;Nichols 2003;Parton and Richards 2003;Bathori et al. 2004;Kirkham and Parton 2005). Due to the advances in endocytosis research, later studies clearly showed that contrary to earlier claims, CPPs utilize endocytic pathways to transport attached cargoes into the cells (Console et al. 2003;Drin et al. 2003;Fittipaldi et al. 2003;Richard et al. 2003). These newer studies confirmed that contrary to early anti-endocytic hypotheses, specific endocytic pathways can be efficiently exploited to deliver biomolecules into the cells (LeCher et al. 2017).
One of the earliest CPPs was a conjugate of the hydrophobic domain of Kaposi's sarcoma fibroblast growth factor signal sequence and the NLS of NF-κB p50 that can enter the cells and block the nuclear import of stress-responsive transcription factors (SRTFs) like NF-κB, AP-1, STAT1, and NFAT in inflammatory conditions (Lin et al. 1995;Torgerson et al. 1998;Zhang et al. 1998;Kolenko et al. 1999;Liu et al. 2000;Letoha et al. 2005a, b, c). This NF-κB NLS-containing conjugate named SN50 was claimed to be internalized via an endocytosis/receptor-independent mechanism (Veach et al. 2004). Cellular internalization was attributed purely to the hydrophobic signal sequence, while the basic NF-κB p50 NLS peptide alone was thought to be non-cell-permeable and thus have no biological effects ). The wild-type (WT) NF-κB p50 NLS peptide (CYVQRKRQKLMP) specifically interacts with the cytoplasmic Rch1/importin-β NLS receptor complex of Jurkat cell extracts, but it was reasoned that without the hydrophobic signal sequence this cationic NLS was unable to enter the cells and inhibit the inducible nuclear import of NF-κB proteins (including p50 and p65) and other SRTFs Boothby 2001). This reasoning seemed inconsistent with the advanced concepts of cationic peptide internalization. As described above, cationic peptides have been widely used to import bioactive cargoes intracellularly, and basic NLS peptides can even transport DNA into the nucleus (Eguchi et al. 2001;Snyder and Dowdy 2001;Akuta et al. 2002;Nakanishi et al. 2003;Arenal et al. 2004;Ahmed 2017;Vedadghavami et al. 2020). Ragin et al. demonstrated that the NLS of the NF-κB p50 subunit is also internalized by cells at 37 °C and facilitates intracellular delivery of attached compounds (Ragin et al. 2002;Ragin and Chmielewski 2004). It is widely established that peptides abundant in arginines and lysines have the unique character to be taken up by cells through endocytic pathways induced by electrostatic binding to polyanionic proteoglycans (Sandgren et al. 2002;Belting 2003;Futaki et al. 2007;Poon and Gariepy 2007;Christianson and Belting 2014;Zhu and Jin 2018). Previously we demonstrated the syndecan-dependent cellular uptake of cationic CPPs (Letoha et al. 2010). Syndecans (SDCs), a family of transmembrane heparan-sulfate proteoglycans (HSPGs), efficiently deliver a wide range of ligands intracellularly by attaching them through their versatile polyanionic heparan-sulfate (HS) sidechains (Christianson and Belting 2014;Afratis et al. 2017). Microbes, growth factors, and other endogenous proteins endowed with cationic heparin-binding sequences can attach to SDCs and enter the cells via SDC-mediated endocytosis (Gallay 2004;Elfenbein and Simons 2013;Favretto et al. 2014;Cagno et al. 2019;Hudak et al. 2019;Letoha et al. 2019;Stow et al. 2020;De Pasquale et al. 2021;Hudak et al. 2021aHudak et al. , b, 2022. Considering the immense evidence on the HSPG-mediated uptake of cationic peptides, we reexamined the cellular internalization and biological activity of the cationic NF-κB p50 NLS unconjugated to any translocating peptide sequence. The peptide was labeled with FITC, and its uptake was investigated in vitro and in vivo. Besides general cell uptake studies, the NLS' internalization was also assessed in SDC-specific cellular assays. Effects of the peptide on NF-κB transcriptional activity were analyzed with various in vitro inflammatory models, including NF-κB reporter gene assays enabling the quantitative assessment of inducible NF-κB activity based on luminescence (Letoha et al. 2005a, b, c;Letoha et al. 2006). The NF-κB inhibitory activity of the peptide was tested in vivo in an experimental model of acute pancreatitis, an inflammatory disorder initiated by the activation of SRTFs, including NF-κB and AP-1 (Gukovsky et al. 2003;Letoha et al. 2005aLetoha et al. , b, c, 2006Letoha et al. , 2007Gukovsky and Gukovskaya 2013;Yu and Kim 2014). Since activated NF-κB is one of the most significant initiators of pancreatic inflammation, CCK-induced acute pancreatitis offered an ideal experimental model to monitor the efficacy of the p50 NLS in inhibiting NF-κB activity in vivo (Gukovsky et al. 1998;Williams et al. 2002;Letoha et al. 2005aLetoha et al. , b, c, 2006Letoha et al. , 2007Gukovsky and Gukovskaya 2013;Huang et al. 2013). The broad inhibitory spectrum of the p50 NLS on NF-κB proteins (i.e., p50 NLS competes for proteins generally involved in nuclear import of NF-κB proteins, including p50 and p65) seemed also beneficial in the complex NF-κB activation pathway of pancreatitis (Boothby 2001;Gukovsky and Gukovskaya 2013;Huang et al. 2013).
Our results confirm that HSPG-, particularly SDC-mediated endocytosis can be utilized for efficient peptide transduction in vitro and in vivo. Thus, the present manuscript undermines the receptor-independent, non-endocytic uptake of the cationic NLS peptide and marks new pathways to be utilized for the intracellular delivery of novel bioactive, cationic peptides. endothelial cells, L929 murine fibroblasts, and RAW 264.7 murine macrophages) showed a gradual increase in fluorescence following the addition of the FITC-labeled NLS peptide at a concentration of 20 μM at 37 °C (Fig. 1). Intracellular fluorescence increased markedly at 30 min of incubation, demonstrating efficient internalization. At 90 min the FITC-labeled p50 NLS peptide appeared in the nuclei where it accumulated. Adding the NLS peptide to cells at 4 °C, a temperature where the rigidity of cellular membranes unable endocytosis, hindered the cellular entry of the peptide, hence demonstrating the endocytic nature of uptake ( Supplementary Fig. S1). Replacing the basic residues with glycines (i.e., CYVQGGGQGLMP, indicated as GlyNLS) also impeded cellular uptake of the NLS, as intracellular fluorescence of cells treated with the FITC-labeled GlyNLS analog remained almost undetectably low even at 90 min of incubation at 37 °C, hence demonstrating the lack of uptake due to the loss of the basic residues ( Supplementary Fig.  S1). Simultaneous cell viability studies showed that neither the NLS nor its Gly analog (GlyNLS) affected cellular viability from concentrations of 1.56 μM to 50 μM (Supplementary Fig. S2).

Flow Cytometric Assessment of the NF-κB p50 NLS
Cellular uptake of the FITC-labeled p50 NLS peptide (NLS; at a concentration of 20 μM) was then quantified with standard flow cytometry. Fluorescence of surface-bound peptides was quenched by adding trypan blue (at a concentration of 0.25% in ice-cold 0.1 M citrate buffer pH 4.0) one minute before flow cytometry hence only the intracellular FITC-labeled NLS was measured (Letoha et al. 2010. After 30 min of incubation, a small increase in the cellular fluorescence of the NLS-treated cells could be detected at 37 °C ( Fig. 2a). At 60 and 90 min, intracellular fluorescence further increased, especially in RAW macrophages (Fig. 2b, c). ATP-depletion (with 0.1% mM sodium azide and 50 mM 2-deoxy-D-glucose) resulted in low intracellular fluorescence, which showed diminished peptide uptake ( Fig. 2a-c). Pretreating the cells with methyl-β-cyclodextrin (MCD) to remove cholesterol from the membrane markedly reduced intracellular fluorescence, suggesting the involvement of the cholesterol-enriched lipid rafts in the cellular internalization of the peptide at 37 °C ( Fig. 2a-c). Exogenous heparan-sulfate (HS; 25 μg/mL) also diminished intracellular fluorescence at 37 °C, indicating the involvement of polyanionic surface proteoglycans in attaching the cationic peptide ( Fig. 2a-c). At 4 °C the cellular membranes become extremely rigid which stops endocytosis. At 4 °C all cells treated with the FITC-labeled peptide exhibited very low cellular fluorescence, providing further evidence of the endocytic nature of NLS uptake (Fig. 2d).

SDCs Mediate the Cellular Internalization of the NF-κB p50 NLS
The contribution of SDCs to several cationic CPPs have already been explored (Letoha et al. 2010;Montrose et al. 2013). Considering the previously obtained data on the HSdependent uptake of the NLS, we studied its uptake in cell lines expressing specific SDC isoforms. K562 cells reportedly exhibit very low endogenous HSPG expression, except for a minimal amount of betaglycan and SDC3 (Shafti-Keramat et al. 2003;Letoha et al. 2010). Due to their low HSPG expression and lack of caveolin-1, the molecular base of caveolae-mediated endocytosis, K562 cells offer an ideal cellular environment to express SDC isoforms and study their functionality without the interference of other HSPGs and caveolae-mediated endocytosis (Parolini et al. 1999;Hudak et al. 2019;Letoha et al. 2019). After creating stable SDC transfectants in K562 cells, the various SDC-expressing clones were selected and standardized according to their HS expression ( Supplementary Fig. S3) (Hudak et al. , 2021b(Hudak et al. , 2022Letoha et al. 2019). Thus stable SDC transfectants with even HS expression were selected and, along with WT K562 cells, incubated with the FITC-labeled NLS peptide for 90 min at 37 °C. Cellular internalization of the fluorescently labeled NLS was assessed with imaging flow cytometry. To remove surface fluorescence due to extracellularly attached fluorescent NLS, peptide-treated cells were  Hudak et al. 2019Hudak et al. , 2021bHudak et al. , 2022. Imaging flow cytometry demonstrated that SDC overexpression increases NLS uptake, especially SDC4, that increased cellular uptake of the FITC-labeled NLS the most ( Fig. 3a-c). Simultaneous cell viability measurements showed that the NLS did not affect cellular viability at the applied concentrations of 20 μM ( Supplementary  Fig. S4).
The NF-κB inhibitory effect of the peptide was then measured in LPS-stimulated murine macrophages. The p50 NLS decreased the LPS-induced luciferase activity of RAW 264.7 macrophages, however, to a smaller extent than in the case of TNF-α-induced L929 fibroblasts (Fig. 4b).

The NF-κB p50 NLS Peptide Ameliorates Acute Experimental Pancreatitis
After demonstrating the efficient intracellular uptake and the NF-κB suppressing the activity of the p50 NLS in vitro, we tested the peptide's anti-inflammatory effects in an animal model of acute pancreatitis, a disease triggered by activated SRTFs including NF-κB.
First, we analyzed whether the peptide could enter the pancreas in vivo. According to the Human Protein Atlas, the pancreas shows a definite SDC4 expression (Uhlen et al. 2005(Uhlen et al. , 2015. Thus, pancreas tissues were dissected from rats 15 min after intraperitoneal (IP) injection of the FITC-labeled p50 NLS peptide or pure PBS (controls). Fluorescent microscopic analysis revealed fluorescent intracellular signals, the characteristic features of endocytosis in the pancreatic samples of the NLS-injected rats (Fig. 5b,c), demonstrating that the NF-κB p50 NLS peptide was internalized in vivo. Contrary to the NLS-injected rats, the pancreas of control animals displayed low and blunt autofluorescence without any distinct intracellular signs of endocytosis ( Fig. 5a-c). Cellular uptake studies also showed the efficient cellular entry of the fluorescently labelled p50 NLS peptide into AR42J pancreatic acinar cells ( Supplementary  Fig. S6). Unlike the p50 NLS peptide, the GlyNLS analog in which the basic residues are replaced with glycines (i.e., CYVQGGGQGLMP) exhibited no sign of cellular uptake into AR42J cells.
After showing its efficient in vivo delivery into the pancreas, the NLS' effects were studied in the experimental model of CCK-induced pancreatitis. Rats receiving 2 × 100 μg/kg body weight of CCK IP exhibited the relevant molecular and histological features of pancreatic inflammation, including intrapancreatic edema and cellular damage (as revealed by the increased pancreatic weight/body weight ratio and serum amylase activity in Fig. 6a). Concentrations of proinflammatory cytokines, TNF-α and IL-6 in the pancreas were also significantly increased (Fig. 6b). Supramaximal CCK doses triggered leukocyte sequestration, raising MPO activities in the pancreas and the lung (Fig. 6c). CCK also induced ROS production. Thus the level of MDA, the measure of lipid peroxidation, was significantly higher compared to controls. Due to increased ROS production, intrapancreatic GSH (that participates in eliminating ROS) was depleted after two injections of CCK (Fig. 6d). Pretreatment with 2 mg/kg of the NF-κB p50 NLS peptide IP 30 min before the first CCK dose improved all these laboratory parameters, thus ameliorating the pancreatitis-inducing effects of CCK ( Fig. 6a-d).
Electrophoretic mobility shift assay (EMSA) performed on nuclear extracts of pancreas samples was utilized to assess the NF-κB inhibitory effect of the peptide. The DNA-binding activity of NF-κB was relatively weak in the untreated controls ( Fig. 7). CCK administration significantly increased the DNAbinding activity of NF-κB, which could be inhibited with the p50 NLS pretreatment. While CCK also induced the degradation of IκB-α, pretreatment with the p50 NLS peptide had no significant effect on CCK-induced degradation of IκB-α ( Supplementary Fig. S7).
Histology revealed that administration of 2 × 100 μg/kg body weight CCK induced acute pancreatitis, which was characterized by microfocal necrosis, vacuolar degeneration, marked edema, inflammatory activity, and stasis (Fig. 8a). Pretreatment with 2 mg/kg of the NF-κB p50 NLS peptide significantly reduced the morphological damage induced by CCK in the pancreas (Fig. 8b). The values for each of the scored parameters are shown in Table 1. Luciferase reporter assays of TNF-α-triggered L929 fibroblasts (a) and LPS-activated RAW 264.7 macrophages (b) with NF-κB-Luc are shown. Controls were treated with 10 U/mL of TNF-α or 30 ng/ mL LPS. The p50 NLS-treated cells were incubated with various peptide concentrations for 30 min before 10 U/mL TNF-α or 30 ng/mL LPS was added. Luciferase activity was analyzed 6 h later. Detected luminescence intensities were normalized to TNF-α or LPS-onlytreated cells as standards. The bars represent the means + SEM of four independent experiments. c Surface ICAM-1 expression as detected with flow cytometry on HMEC-1 cells pretreated with the p50 NLS for 30 min before TNF-α (10 U/mL) incubation for 6 h. Detected ICAM-1 expression values were normalized to TNF-α-onlytreated cells as standards. The bars represent the means + SEM of four independent experiments Fig. 5 The pancreas internalizes the NF-κB p50 NLS in vivo. The in vivo internalization studies were carried out by injecting 20 nM/kg body weight of FITC-labeled NF-κB p50 NLS or PBS (Ctrl) IP into male Wistar rats. The images show pancreas samples taken from rats 15 min after injecting PBS (a) or the FITC-labeled NLS peptide (b) IP. Images represent three independent studies. Scale bar = 50 μm. c BioTek Gen5 Software was utilized to assess fluorescence intensity of pancreatic samples. Two samples from each group from three independent studies were analyzed. Detected fluorescence intensities were normalized to PBS-treated controls. The bars represent the mean + SEM of six samples from three independent experiments. Statistical significance vs. Ctrl was assessed with ANOVA. ***p < 0.001 Fig. 6 The NF-κB p50 NLS peptide improves the laboratory markers of acute pancreatitis in vivo. Acute pancreatitis was induced with 2 × 100 μg/kg of CCK IP in male Wistar rats. p50 NLS peptidetreated animals received 2 mg/kg IP of the p50 NLS 30 min before the first CCK dose. Figure a shows pancreatic weight/body weight ratio and serum amylase activity, b shows intrapancreatic TNF-α and IL-6 levels, c shows pancreatic and lung MPO activity and d shows pancreatic MDA and GSH levels. Means + SEM of 10 animals in each group are shown. Light gray bars represent controls (receiving 3 × 0.5 mL of PBS IP), black bars represent Group CCK (animals receiving 2 × 100 μg/kg of CCK IP) and dark gray bars represent Group NLS + CCK (animals treated with 2 mg/kg of p50 NLS IP 30 min before the first injection of CCK). *p < 0.05 vs Group CCK; **p < 0.01 vs Group CCK

Discussion
As proteoglycan-mediated endocytic uptake of cationic peptides is now widely established, it is time to reconsider our views about the internalization of biomolecules. Our study demonstrates that basic nuclear localization signal (NLS) of the NF-κB p50 subunit-without any cell-transporter motif attached-can be efficiently internalized through proteoglycan-mediated endocytic pathways and retain its reported biological activity to inhibit NF-κB's nuclear translocation and transcriptional activity.
Considering current knowledge on proteoglycan-mediated peptide transduction, it is unsurprising that a cationic 12-mer NLS peptide can enter the cells via endocytosis. Endocytosis is a complex mechanism that involves clathrindependent and independent pathways (Kumari et al. 2010). Clathrin-independent endocytosis includes phagocytosis, constitutive pinocytotic pathways, and endocytosis mediated by caveolae and glycolipid rafts (Nichols and Lippincott-Schwartz 2001;Nichols 2003;Parton and Richards 2003;Kirkham and Parton 2005). Contrary to classic clathrindependent endocytosis, caveolar or lipid raft-mediated internalization can avoid the lysosomes and hence the degradation of internalized molecules (Bathori et al. 2004;Kiss and Botos 2009;Sousa de Almeida et al. 2021). As recycling and internalization of proteoglycans occur through lipid rafts, thus by attaching to polyanionic cell surface proteoglycans, cationic peptides can utilize lipid raft-mediated pathways to enter the cells (Belting 2003;Christianson and Belting 2014). The importance of binding to polyanionic heparan sulfate proteoglycans and entering the cells by lipid-raftmediated endocytosis was clearly confirmed in our flow cytometric uptake experiments when incubating the cells with HS or MCD markedly decreased internalization of the NLS peptide. The replacement of basic residues with glycine Fig. 7 The NF-κB p50 NLS peptide inhibits NF-κB transcription activity in acute pancreatitis in vivo. a A representative EMSA showing DNA-binding activity of NF-κB in pancreatic samples. b Intensities of NF-κB bands were densitometrically quantified relative to untreated controls (i.e., normal pancreas). Values presented are means + SEM, n = 10 animals/group. Light gray bars represent controls (receiving 3 × 0.5 mL PBS IP), black bars represent Group CCK (animals receiving 2 × 100 μg/kg of CCK IP) and dark gray bars represent Group NLS + CCK (animals treated with 2 mg/kg of p50 NLS IP 30 min before the first injection of CCK). *p < 0.05 vs Group CCK; **p < 0.01 vs Group CCK Fig. 8 Effect of the NF-κB p50 NLS on pancreatic morphological damage in CCK-induced pancreatitis. a A representative pancreatic sample from the Group CCK (animals treated with 2 × 100 μg/kg of CCK IP) showing marked edema, inflammatory activity, microfocal necrosis (HE × 100); and microfocal necrosis, vacuolar degeneration in the acinar cells (insert: HE × 250). Scale bar = 50 μm. b A representative pancreatic sample from animals receiving p50 NLS pretreatment before CCK (i.e., Group NLS + CCK): milder edema, milder acinar degeneration, and vacuolization (HE × 100 and HE × 250). Scale bar = 50 μm Table 1 Effects of the NLS on the histologic parameters in CCKinduced acute pancreatitis Means + SEM of 10 animals in each group are shown *p < 0.05 vs Group CCK; **p < 0.01 vs Group CCK

Controls
Group CCK Group NLS + CCK Edema 0.1 ± 0.067** 0.9 ± 0.067 0.65 ± 0.076* Vascular change 0.1 ± 0.067** 0.7 ± 0.082 0.45 ± 0.05* Inflammation 0** 0.6 ± 0.07 0.35 ± 0.076* Acinar necrosis 0** 0.75 ± 0.083 0.45 ± 0.09* Calcification 0 0.125 ± 0,065 0 Fat necrosis 0* 0.167 ± 0.071 0.05 ± 0.05 also abolished peptide uptake, highlighting the paramount role of electrostatic interactions in peptide uptake. Both ATP-depletion or low temperature blocked peptide entry, further providing evidence on the energy-dependent and temperature-sensitivite endocytic uptake mediated by polyanions of the cholesterol-enriched lipid rafts. Proteoglycans comprise a heterogeneous group of proteins substituted with linear polysulfated and, thereby, highly negatively charged glycosaminoglycan polysaccharides (e.g. heparan sulfate) (Iozzo 2001;Letoha et al. 2006;Sarrazin et al. 2011). Cell surface proteoglycans bind a multitude of ligands and influence cellular physiology and pathology, including cytokine-signaling and inflammation (Letoha et al. 2006;Billings and Pacifici 2015;O'Callaghan et al. 2018;El Masri et al. 2020). Among cell surface proteoglycans, the SDC family of transmembrane HSPGs act as molecular ferries by attaching cationic proteins and delivering them intracellularly (Letoha et al. 2010(Letoha et al. , 2021a(Letoha et al. , 2021b(Letoha et al. , 2022Christianson and Belting 2014;Hudak et al. 2019). SDC-mediated endocytosis occurs independently of clathrin and caveolin but in a lipid raft-dependent manner: ligands induce clustering and redistribution of SDCs to lipid rafts and stimulate the lipid raft-dependent endocytosis of the SDC-ligand complex (Payne et al. 2007;Szilak et al. 2013;Christianson and Belting 2014;Hudak et al. 2019). Besides their endocytic activity, SDCs are also heavily involved in cell signaling and transmit signals from the cell exterior to the cytoplasm (Tkachenko et al. 2005;Couchman et al. 2015;Afratis et al. 2017). Thus, the SDC-mediated uptake of the p50 NLS peptide can explain its efficient internalization and the maintenance of its ability to inhibit NF-κB-driven inflammatory pathways, as demonstrated by the inhibition of NF-κB activities in vitro and in vivo. Among SDCs, the p50 NLS peptide showed the highest affinity towards SDC4, the ubiquitously expressed member of the SDC4 family (Letoha et al. 2010Keller-Pinter et al. 2021). Still, the overexpression of the other SDC isoforms also increased the cellular uptake of the peptide markedly. SDCs enables the entry of the peptide into a wide array of SDC-expressing cells, including, but not limited to macrophages and pancreatic acinar cells, two key cells types whose Ca2 + overload initiates acute pancreatitis (Uhlen et al. 2015;Gryshchenko et al. 2021;Petersen et al. 2021). During acute pancreatitis, excessive Ca2 + signal generation also occurs in pancreatic stellate cells, a cell type with pronounced SDC4 expression (Chronopoulos et al. 2020;Petersen et al. 2021).
In live rats, the peptide rapidly entered the pancreas, an organ exhibiting pronounced SDC expression (Uhlen et al. 2015). The observed rapidity of the peptide's in vivo internalization is also advantageous in the experimental model of CCK-induced acute pancreatitis, where NF-κB activation and nuclear translocation peak 30 min after CCK stimulation (Gukovsky et al. 1998;Letoha et al. 2006). The p50 NLS' reported broad inhibitory spectrum on the nuclear import NF-κB proteins (i.e., intracellularly delivered p50 NLS is not specific for p50, but it also blocks the nuclear import of p65 and other SRTFs) also offers significant therapeutic benefits in an acute inflammatory disorder regulated by complex NF-κB activation pathways Boothby 2001;Wu et al. 2020). The performed EMSA showed that p50 NLS pretreatment could suppress CCK-induced nuclear translocation and DNA-binding activity of NF-κB. NF-κB inhibitory activity of the NLS peptide resulted in improved parameters of pancreatitis. Thus, pretreatment with the NF-κB p50 NLS peptide ameliorated CCK-induced cellular damage, edema and neutrophil sequestration both within the pancreas and lung. Moreover, pretreatment with the p50 NLS decreased proinflammatory cytokines and ROS production in the pancreas. Histopathological evaluation of pancreas samples also confirmed improvements due to p50 NLS treatment. Thus, the cationic NLS peptide could also preserve its NF-κB-inhibitory and anti-inflammatory effects in vivo.
In summary, our study demonstrates the efficient intracellular delivery of a cationic NLS peptide to inhibit NF-κBdependent inflammatory pathways and provides preclinical proof of concept on the efficient utilization of SDC-mediated peptide transduction to modulate acute inflammation of the pancreas.

Peptide Synthesis and Labeling
The NF-κB p50 NLS peptide (CYVQRKRQKLMP) and its Gly analog (GlyNLS, CYVQGGGQGLMP) were synthesized in solid phase by standard methodology as described previously . For the uptake experiments, the peptides were labeled with fluorescein isothiocyanate (FITC; cat. no. 46950; Sigma-Aldrich, Darmstadt, Germany) as described by Fulop et al. (2001). CCK was prepared with the method of Penke et al. (1984).

Cell Viability Measurements
The effect of the applied peptides (NLS and its GlyNLS analog) on cell viability was assessed with the EZ4U cell proliferation assay (Biomedica Gmbh, Vienna, Austria, cat. no. BI-5000), according to the instructions of the manufacturer (Hudak et al. 2021b). Absorbance was measured with a BioTek Cytation 3 multimode microplate reader.

Confocal Laser Scanning Microscopy
Internalization of the FITC-labeled p50 NLS peptide into L929, RAW 264.7 and HMEC-1 cells was visualized by confocal laser scanning microscopy. Cells were preincubated in Opti-MEM (cat. no. 31985062; Thermo Fisher Scientific, Waltham, MA, USA) at either 37 or 4 °C for 30 min before incubation with the peptides (Letoha et al. 2006). The peptide solution was prepared at a concentration of 20 μM in Opti-MEM by diluting a 1 mM stock solution of peptide in phosphate-buffered saline (PBS) (Letoha et al. 2005a, b, c). After various amounts of time (30, 60, 90 min) at 37 and 0 °C, the cells were rinsed three times with ice-cold PBS, and fluorescence distribution was immediately analyzed on an Olympus FV1000 confocal laser scanning microscope. Excitation was obtained with an Argon ion laser set at 488 nm for FITC excitation and the emitted light was filtered with an appropriate long-pass filter (514 nm). Sections presented were taken approximately at the mid-height level of the cells. Photomultiplier gain and laser power were identical within each experiment. Cell viability was routinely determined by using trypan blue exclusion tests (Letoha et al. 2005a, b, c).

Flow Cytometry Analysis of Peptide Uptake
L929 fibroblasts, RAW 264.7 macrophages, and HMEC-1 cells were used to quantify the cellular internalization of the fluorescent peptide. 6 × 10 5 cells/mL in Opti-MEM were incubated with the FITC-labeled peptide (at a concentration of 20 μM) at 37 and 4 °C for 30, 60 and 90 min, respectively.
The cells were washed twice and resuspended in 0.5 mL of physiological saline. Equal volumes of this suspension and a stock solution of trypan blue (cat. no: T6146-5G; Merck KGaA, Darmstadt, Germany; 500 μg/mL dissolved in icecold 0.1 M citrate buffer at pH 4.0) were allowed to mix for 1 min before FACS analyses. In this way, sample pH was lowered to pH 4.0, thereby optimizing the quenching effect of trypan blue (Sahlin et al. 1983). Cellular uptake was measured with flow cytometry using a FACScan (Becton Dickinson, Franlin Lakes, NJ, USA). A minimum of 10,000 events per sample was analyzed. The viability of the cells was determined by appropriate gating in a forward-scatteragainst-side-scatter plot to exclude dead cells, debris, and aggregates (Letoha et al. 2005a, b, c).
To investigate the involvement of cholesterol-rich membrane domains (lipid rafts) in peptide uptake, cells were pretreated with 5 mM methyl-β-cyclodextrin (MCD; Sigma-Aldrich, cat.no: C4555-5G) for 60 min at 37 °C and then treated as mentioned above (Letoha et al. 2005a, b, c). To study the role of polyanionic cell-surface proteoglycans the cells were incubated with the FITC-labeled peptides in the presence of HS (25 μg/mL; Sigma-Aldrich; cat. no. H4777-1MG) in Opti-MEM and then processed as usual for the FACS analyses.
To block endocytosis, cells were either incubated at 4 °C or their cellular ATP pools were depleted. For experiments at 4 °C, the cells were maintained for 30 min on ice before peptide incubation and throughout the experiments (Letoha et al. 2005a, b, c). To induce ATP depletion, the cells were incubated with 0.1% sodium azide (Sigma-Aldrich; cat.no. S2002-5G) and 50 mM 2-deoxy-D-glucose (Sigma-Aldrich; cat.no. D8375-1G) in Opti-MEM for 60 min before the addition of peptides at 37 °C (Fischer et al. 2004).

Imaging Flow Cytometry Analysis of Peptide Uptake
WT K562, SDC transfectants or AR42J cells were utilized to quantify the internalization of the FITC-labeled p50 NLS peptide. Briefly, 6 × 10 5 cells/mL in DMEM/F12 medium were incubated with FITC-labeled p50 NLS or its Gly analog (at a concentration of 20 μM) for 90 min (15 min for AR42J) at 37 °C (Letoha et al. 2005a, b, c). After incubation with the, the cells were trypsinized (with the method described by Nakase et al.) to remove the extracellularly attached peptides from the cell surface Hudak et al. 2019Hudak et al. , 2021bHudak et al. , 2022. The cells were then rinsed three times with PBS containing 1% BSA and progressed towards flow cytometry. Cellular uptake was then measured with flow cytometry using an Amnis FlowSight imaging flow cytometer (Amnis Corporation, Seattle, WA, USA). A minimum of 10,000 events per sample was analyzed. Appropriate gating in a forward-scatter-against-side-scatter plot was utilized to exclude cellular debris and aggregates.
Fluorescence analysis was conducted with the Amnis IDEAS analysis software (Hudak et al. 2021b).

ICAM-1 Expression
ICAM-1 expression of HMEC-1 cells, grown on microplates (Corning Life Sciences), was conducted as described previously (Letoha et al. 2005a(Letoha et al. , b, c, 2006. 3 × 10 4 cells/well in HE-SFM 2% FCS were exposed to various concentrations (3.13 to 50 uM) of the p50 NLS peptide for 30 min. Thirty minutes later the cells were treated with TNF-α (10 U/mL in 100 μL of the above medium per well). After 6 h of incubation with TNF-α, the cells were trypsinized, washed and resuspended in 10% FCS, then vortexed for 5 min at 2000 rpm, resuspended in PBS and vortexed again for 5 min. Then the medium was removed and the cells were incubated in PBS with the FITC-conjugated monoclonal mouse anti-human ICAM-1 antibody (5-10 µg/mL; cat. no. BMS108FI; Thermo Fischer Scientific) for 30 min on ice. After two washes and fixation with 2% paraformaldehyde, while being vortexed, the samples were analyzed by flow cytometry using the FACScan flow cytometer and the CellQuest analysis program (Becton Dickinson). Control cells received only TNF-α treatment (Letoha et al. 2006). The viability of the cells was determined by concurrent propidium iodide (10 µg/mL; Sigma-Aldrich; cat. no. P4170-250MG) staining and appropriate gating in a forward-scatter-againstside-scatter plot to exclude dead cells, debris, and aggregates (Letoha et al. 2005a, b, c).

In Vivo Uptake Experiments
In vivo uptake studies in male Wistar rats (provided by the Animal Center of the Biological Research Center) weighing 250-300 g were carried out as described previously (Letoha et al. 2005a(Letoha et al. , b, c, 2006(Letoha et al. , 2007. The animals were kept at a constant room temperature with a 12 h light-dark cycle and were allowed free access to water and standard laboratory chow (Innovo Kft., Isaszeg, Hungary). All animal experiments were performed according to national and institutional ethical guidelines. The animal study protocol was approved by the Institutional Animal Ethics Committee of the Biological Research Centre and by the National Scientific Ethical Committee on Animal Experimentation (protocol code XVI./04714/001/2006., approved on 08 March 2006) and complied with the European Communities Council Directive of 24 November 1986 (86/609/ EEC). Six animals were intraperitoneally (IP) injected with 20 nM/kg of the FITC-labeled p50 NLS peptide (in 0.5 mL PBS). Control animals (n = 6) received IP injections of 0.5 mL PBS. Rats were anesthetized (with pentobarbital sodium 50 mg/kg IP) and killed 15 min after the injections by exsanguinations through the abdominal aorta. Pancreas tissues were harvested and frozen in His-toPrep media (cat. no. SH75-125D; Thermo Fisher Scientific). Sections (10 to 50 μm) were cut on a cryostat and analyzed with fluorescence microscopy (BioTek Cytation 3). The fluorescence signals detected were measured with BioTek Gen5 Software. The animal study is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).

CCK-Induced Pancreatitis
CCK-induced pancreatitis utilizing various experimental groups of rats (each group contained 10 animals) was carried out as described previously (Letoha et al. 2005a(Letoha et al. , b, c, 2006(Letoha et al. , 2007. The rats were fasted for 16 h then acute pancreatitis was induced by injecting 100 μg/kg body weight of CCK (dissolved in PBS) IP twice at an interval of 1 h ("Group CCK"). The p50 NLS pretreated group ("Group NLS + CCK") received 2 mg/kg body weight of the NF-κB p50 NLS peptide (in PBS) IP 30 min before the first injection of CCK. Control rats received 3 × 0.5 mL PBS IP instead of CCK or the NF-κB p50 NLS. Anesthetized (pentobarbital sodium; 50 mg/kg IP; Sigma Aldrich; cat. no. P3761) rats were killed by exsanguinations through the abdominal aorta 4 h after the first CCK injection (Letoha et al. 2005a(Letoha et al. , b, c, 2006(Letoha et al. , 2007. The pancreas and lungs were quickly removed, cleaned of fat and lymph nodes, weighed, frozen in liquid nitrogen and stored at − 80 °C until use (Letoha et al. 2005a(Letoha et al. , b, c, 2006(Letoha et al. , 2007.

Molecular Markers of Acute Pancreatitis
Molecular markers of acute pancreatitis were analyzed as described previously (Letoha et al. 2005a(Letoha et al. , b, c, 2006(Letoha et al. , 2007. The pancreatic weight/body weight ratio was utilized to evaluate the degree of pancreatic edema. The serum levels of amylase were determined by a colorimetric kinetic method (cat. no. D96569; Dialab, Vienna, Austria). All blood samples were centrifuged at 2500×g for 20 min. Tumor necrosis factor-α (TNF-α) and IL-6 concentrations were measured in the pancreatic cytosolic fractions with ELISA kits (cat. no. ERA57RB and ERA32RB; all Thermo Fisher Scientific) according to the manufacturers' instructions. As a marker of tissue leukocyte infiltration, pancreatic and lung MPO activity was assessed by Kuebler et al. (Kuebler et al. 1996). Pancreatic MDA level was measured after the reaction with thiobarbituric acid, according to the method of Placer et al., and was also corrected for the protein content of the tissue (Placer et al. 1966). GSH level was determined spectrophotometrically with Ellman's reagent (Sedlak and Lindsay 1968).

Histological Evaluation of CCK-Induced Acute Pancreatitis
Histological evaluation of CCK-induced acute pancreatitis was carried out as described previously (Letoha et al. 2005a(Letoha et al. , b, c, 2006(Letoha et al. , 2007. A portion of the pancreas was fixed in 8% neutral formaldehyde solution (Sigma-Aldrich; cat. no. 47608) and subsequently embedded in paraffin (Sigma-Aldrich; cat. no. P3558). Sections were cut at 4 μm thickness and stained with hematoxylin and eosin (cat. no. ab245880; Abcam, Waltham, MA, USA). The slides were coded and read for the traditional histological markers of pancreatic tissue injury by two independent observers who were blind to the experimental protocol. They used the scoring system of Hughes et al. for the evaluation of acute pancreatitis (Hughes et al. 1996). Thus, semiquantitative grading of interstitial edema (0-1), vascular changes (0-2), inflammation (0-1), acinar necrosis (0-2) calcification (0-0.5) and fat necrosis (0-0.5) of the pancreas samples was evaluated in each animal (described in more details in Table 2).

Statistical Analysis
Results are expressed as means + standard error of the mean (SEM). Differences between experimental groups were evaluated by using one-way analysis of variance (ANOVA). Values of p < 0.05 were accepted as significant Letoha et al. 2019).

Conflicts of interest The authors declare no conflicts of interest.
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