Pyrrolidine Dithiocarbamate Suppresses Cutibacterium acnes-Induced Skin Inflammation.

Cutibacterium acnes (C. acnes), a Gram-positive anaerobic bacterium, proliferates in hair follicles and pores and causes inflammation in the skin of young people. The rapid growth of C. acnes triggers macrophages to secrete proinflammatory cytokines. Pyrrolidine dithiocarbamate (PDTC) is a thiol compound that exerts antioxidant and anti-inflammatory effects. Although the anti-inflammatory function of PDTC in several inflammatory disorders has been reported, the effect of PDTC on C. acnes-induced skin inflammation remains unexplored. In the present study, we examined the effect of PDTC on C. acnes-induced inflammatory responses and determined the mechanism by using in vitro and in vivo experimental models. We found that PDTC significantly inhibited the expression of C. acnes-induced proinflammatory mediators, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and NOD-like receptor (NLR) pyrin domain-containing 3 (NLRP3), in mouse-bone-marrow-derived macrophage (BMDM) cells. PDTC suppressed C. acnes-induced activation of nuclear factor-kappa B (NF-κB), which is the major transcription factor for proinflammatory cytokine expression. In addition, we found that PDTC inhibited caspase-1 activation and IL-1ß secretion through suppressing NLRP3 and activated the melanoma 2 (AIM2) inflammasome but not the NLR CARD-containing 4 (NLRC4) inflammasome. Moreover, we found that PDTC improved C. acnes-induced inflammation by attenuating C. acnes-induced IL-1ß secretion in a mouse acne model. Therefore, our results suggest that PDTC has potential therapeutic value for the amelioration of C. acnes-induced skin inflammation.

MAP kinase/ERK kinase 1 (MEK1) phosphorylates regulator of calcineurin 1 (RCAN1) to regulate neuronal differentiation.

Regulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (DSCR) on human chromosome 21 and is related to the Down syndrome (DS) phenotype. To identify a novel binding partner of RCAN1, we performed yeast two-hybrid screening and identified mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) as a partner. MEK1 was able to bind and phosphorylate RCAN1 in vitro and in vivo. MEK1-dependent RCAN1 phosphorylation caused an increase in RCAN1 expression by increasing the protein half-life. Nerve growth factor (NGF)-dependent activation of the MEK1 pathway consistently induced RCAN1 expression. Moreover, we found that RCAN1 overexpression inhibited NGF-induced neurite outgrowth and expression of neuronal marker genes, such as growth cone-associated protein 43 (GAP43) and synapsin I, via inhibition of MEK1-ERK1/2 pathways. Our findings provide evidence that MEK1-dependent RCAN1 phosphorylation acts as an important molecular mechanism in the control of neuronal differentiation.

Laurus nobilis leaf extract controls inflammation by suppressing NLRP3 inflammasome activation.

Laurus nobilis Linn. (Lauraceae), commonly known as Bay, has been used as a traditional medicine in the Mediterranean and Europe to treat diverse immunological disorders. Although the effects of L. nobilis on immunosuppression have been reported, the detailed underlying mechanism remains unclear. In this study, to elucidate the anti-inflammatory mechanism of L. nobilis, we examined the effect of L. nobilis leaf extract on inflammasome activation in mouse bone marrow-derived macrophages. L. nobilis leaf extract inhibited NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation, which was associated with caspase-1 activation, interleukin-1ß secretion, and apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome complex formation. We also observed that 1,8-cineole, the major component of L. nobilis extract, consistently suppressed NLRP3 inflammasome activation. Furthermore, L. nobilis leaf extract attenuated the in vivo expression of proinflammatory cytokines in an acute lung injury mouse model. Our results provide the first evidence that L. nobilis leaf extract modulates inflammatory signaling by suppressing inflammasome activation.

Suppression of Propionibacterium acnes-Induced Skin Inflammation by Laurus nobilis Extract and Its Major Constituent Eucalyptol.

Acne is an inflammatory skin disorder in puberty with symptoms including papules, folliculitis, and nodules. Propionibacterium acnes (P. acnes) is the main anaerobic bacteria that cause acne. It is known to proliferate within sebum-blocked skin hair follicles. P. acnes activates monocytic cell immune responses to induce the expression of proinflammatory cytokines. Although the anti-inflammatory function of the Laurus nobilis (L. nobilis) extract (LNE) on several immunological disorders have been reported, the effect of LNE in P. acnes-mediated skin inflammation has not yet been explored. In the present study, we examined the ability of the LNE to modulate the P. acnes-induced inflammatory signaling pathway, and evaluated its mechanism. LNE significantly suppressed the expression of P. acnes-mediated proinflammatory cytokines, such as IL-1ß, IL-6, and NLRP3. We also found that LNE inhibited the inflammatory transcription factor NF-κB in response to P. acnes. In addition, eucalyptol, which is the main constituent of LNE, consistently inhibited P. acnes-induced inflammatory signaling pathways. Moreover, LNE significantly ameliorated P. acnes-induced inflammation in a mouse model of acne. We suggest for the first time that LNE hold therapeutic value for the improvement of P. acnes-induced skin inflammation.

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Targets Down Syndrome Candidate Region 1 (DSCR1/RCAN1) to control Neuronal Differentiation.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP.

PKA regulates calcineurin function through the phosphorylation of RCAN1: identification of a novel phosphorylation site.

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression.

Caspase-3 targets pro-interleukin-1ß (IL-1ß) to restrict inflammation.

The interleukin (IL)-1 family of cytokines plays a pivotal role in immune responses. Among the members of IL-1 family, IL-1ß is synthesized as an inactive precursor (pro-IL-1ß) and becomes active upon cleavage, which is typically facilitated by inflammasomes through caspase-1. In our research, we explored the potential role of caspase-3 in the cleavage of pro-IL-1ß and found that caspase-3 cleaves pro-IL-1ß, specifically at Asp26. Moreover, we found that in the absence of caspase-3 cleavage, the release of active IL-1ß via the inflammasome is increased. Our study introduces pro-IL-1ß as a new substrate for caspase-3 and suggests that caspase-3-mediated cleavage has the potential to suppress IL-1ß-mediated inflammatory responses.

Peiminine Exerts Its Anti-Acne Effects by Regulating the NF-κB Pathway.

Peiminine is the main natural alkaloid compound extracted from the Chinese herb Fritillaria. Although peiminine is known for its antioxidant and anti-inflammatory effects in conditions such as mastitis and arthritis, its impact on inflammation induced by Cutibacterisum acnes (C. acnes) has not been explored. The aim of this study was to investigate the effect of peiminine on C. acnes-induced inflammatory responses in the skin and to identify the underlying mechanism involved. We discovered that peiminine inhibits the C. acnes-induced expression of inflammatory mediators such as pro-interleukin-1ß (pro-IL-1ß), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in mouse bone marrow-derived macrophages (BMDMs). Peiminine suppressed the activation of nuclear factor-kappa B (NF-κB) without affecting the activation of mitogen-activated protein kinase (MAPK) pathways such as JNK, ERK, and p38 MAPK. In addition, we found that peiminine suppressed inflammatory cytokine expression and ameliorated histological symptoms in C. acnes-induced mouse skin. Our study is the first to provide evidence that peiminine has an inhibitory effect on acne, and it points toward the potential of incorporating peiminine into cosmetic and pharmaceutical formulations for acne treatment.

CREB-mediated Bcl-2 expression contributes to RCAN1 protection from hydrogen peroxide-induced neuronal death.

Regulator of calcineurin 1 (RCAN1) is located on the Down syndrome critical region (DSCR) locus in human chromosome 21. In this study, we investigated the functional role of RCAN1 in the reactive oxygen species (ROS)-mediated neuronal death signaling. We found that RCAN1 was able to protect the cells from H(2)O(2) -induced cytotoxicity. The expression of RCAN1 caused an inhibition of the H(2)O(2) -induced activation of mitogen-activated protein kinases (MAPKs) and AP-1. In contrast, RCAN1 significantly enhanced the activity of cAMP response element-binding protein (CREB). Furthermore, RCAN1 induced the expression of the CREB target gene, Bcl-2. Consistently, knockdown of endogenous RCAN1 using shRNA down regulated the phosphorylation of CREB and the expression of Bcl-2, which protects the cells from H(2)O(2) -induced cytotoxicity. Our data provide a new mechanism for the cytoprotective function of RCAN1 in response to oxidant-induced apoptosis.

The regulator of calcineurin 1 (RCAN1/DSCR1) activates the cAMP response element-binding protein (CREB) pathway.

cAMP response element-binding protein (CREB) is one of the best known transcription factors in the development and function of the nervous system. In this report, we found that the regulator of calcineurin 1 (RCAN1), which is overexpressed in the brain of patients with Down syndrome, increased the phosphorylation of CREB and cAMP response element-mediated gene transcription in response to the activation of the intracellular cAMP pathway. Furthermore, we found that the increased activation of CREB signaling by RCAN1 depended on the ability of RCAN1 to inhibit calcineurin activity. Our data provide the first evidence that RCAN1 acts as an important regulatory component in the control of CREB signaling.

Protein kinase A phosphorylates Down syndrome critical region 1 (RCAN1).

The Down syndrome critical region 1 (DSCR1) gene encodes a regulator of the calcineurin 1 (RCAN1) protein, and the elevated levels of RCAN1 are associated with Alzheimer's disease (AD) and Down syndrome (DS). In this report, we found that protein kinase A (PKA) was able to phosphorylate RCAN1 in vitro and in vivo. In addition, we found that the phosphorylation of RCAN1 by PKA caused an increase of RCAN1 expression by increasing of the half-life of the protein. Consistently, the pharmacological inhibition of intracellular PKA using H-89 and the knockdown of the endogenous PKA catalytic subunit with siRNA decreased the expression of RCAN1. Furthermore, the phosphorylation of RCAN1 by PKA enhanced the inhibitory function of RCAN1 on calcineurin-mediated gene transcription. Our data provide the first evidence that PKA acts as an important regulatory component in the control of RCAN1 function through phosphorylation.

Cynanchi atrati and Its Phenolic Constituent Sinapic Acid Target Regulator of Calcineurin 1 (RCAN1) to Control Skin Inflammation.

Atopic dermatitis (AD) is a common inflammatory skin disorder, and numerous pharmacological approaches are employed to reduce symptoms. Natural products of plant-derived materials have been accepted as complementary therapy for the treatment of a wide range of inflammatory diseases. Cynanchi atrati (CA) is an oriental medicinal herb used in the treatment of acute urinary infection, febrile diseases, and laryngopharyngitis. However, the role of CA root extract in skin inflammation such as AD has not been explored yet. In this study, we examined the possible effect of CA root extract on skin inflammation and evaluated the underlying signaling mechanism using in vitro and in vivo modeling systems. Raw264.7 macrophages were used for in vitro experiments, and an oxazolone-induced AD mouse model was used to evaluate in vivo effects. CA extract significantly inhibited the expression levels of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in RAW264.7 macrophages. The CA root extract mediated suppression of pro-inflammatory cytokine expression and was associated with the decreased nuclear factor kappa B (NF-κB) gene transcriptional activation. Moreover, CA root extract attenuated the in vivo expression of IL-6 and tumor necrosis factor-α (TNF-α) and ear swelling in the AD mouse models. We also observed that the inhibitory effect of CA root extract on skin inflammation was accompanied by the upregulation of calcineurin 1 (RCAN1) expression, which functions in the inflammatory pathways by suppressing NF-κB signaling. We consistently observed that the immunosuppressive effect of CA root extract in AD was significantly perturbed in the RCAN1 knockout mice. In addition, we isolated a phenolic acid compound, sinapic acid (SA), from the CA root extract and found that SA consistently exerted an immunosuppressive effect in RAW264.7 macrophages by inducing RCAN1 expression. Our results provide the first evidence that CA root extract and its phenolic acid constituent, SA, modulate NF-κB signaling pathways by inducing RCAN1 expression in the skin inflammation process. Thus, we suggest that CA root extract has a therapeutic value for the treatment of AD by targeting endogenous immune regulators.

Cynanoside F Controls Skin Inflammation by Suppressing Mitogen-Activated Protein Kinase Activation.

Atopic dermatitis (AD) is a chronic inflammatory skin disease accompanied by severe itching and dry skin. Currently, the incidence of AD due to excessive activation of immune cells by various environmental factors is increasing worldwide, and research on inflammatory response inhibitors with fewer side effects is continuously needed. Cynanoside F (CF) is one of the pregnane-type compounds in the root of Cynanchum atratum, an oriental medicinal herb that has been shown to have antioxidant, antitumor, and anti-inflammatory effects. Although CF has been isolated as a component in Cynanchum atratum, the scientific role of CF has not yet been explored. In this study, we evaluated the effect of CF on AD and revealed the mechanism using in vitro and in vivo experimental models. CF significantly reduced lipopolysaccharide (LPS)-induced protein expression levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2), which are important proinflammatory mediators in the RAW264.7 macrophage cell line. CF did not inhibit the nuclear factor-kappa B (NF-κB) signaling activated by LPS but significantly reduced the phosphorylation of mitogen-activated protein kinases (MAPKs), such as p38 MAPK, JNK, and ERK. CF consistently inhibited the activity of the activator protein-1 (AP-1) transcription factor, a downstream molecule of MAPK signaling. In addition, in an experiment using an oxazolone-induced AD mouse model, the CF-treated group showed a marked decrease in epidermal thickness, the number of infiltrated mast cells, and the amount of histamine. The mRNA levels of IL-1ß, interleukin-4 (IL-4), and thymic stromal lymphopoietin (TSLP) were consistently lowered in the group treated with CF. Moreover, the phosphorylation of c-Jun and c-Fos protein levels, which are the AP-1 components, were lowered in the skin tissues of CF-treated mice. These results provide the first evidence that CF has an inhibitory effect on AD and suggest the possibility of CF being developed as a potential therapeutic agent for AD.

Sinapic Acid Controls Inflammation by Suppressing NLRP3 Inflammasome Activation.

A natural phenolic acid compound, sinapic acid (SA), is a cinnamic acid derivative that contains 3,5-dimethoxyl and 4-hydroxyl substitutions in the phenyl ring of cinnamic acid. SA is present in various orally edible natural herbs and cereals and is reported to have antioxidant, antitumor, anti-inflammatory, antibacterial, and neuroprotective activities. Although the anti-inflammatory function of SA has been reported, the effect of SA on the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome has not been explored. In the present study, to elucidate the anti-inflammatory mechanism of SA, we examined whether SA modulates the NLRP3 inflammasome. We found that SA blocked caspase-1 activation and IL-1ß secretion by inhibiting NLRP3 inflammasome activation in bone marrow-derived macrophages (BMDMs). Apoptosis-associated speck-like protein containing CARD (ASC) pyroptosome formation was consistently blocked by SA treatment. SA specifically inhibited NLRP3 activation but not the NLRC4 or AIM2 inflammasomes. In addition, SA had no significant effect on the priming phase of the NLRP3 inflammasome, such as pro-IL-1ß and NLRP3 inflammasome expression levels. Moreover, we found that SA attenuated IL-1ß secretion in LPS-induced systemic inflammation in mice and reduced lethality from endotoxic shock. Our findings suggest that the natural compound SA has potential therapeutic value for the suppression of NLRP3 inflammasome-associated inflammatory diseases.

Pyrrolidine dithiocarbamate (PDTC) inhibits inflammatory signaling via expression of regulator of calcineurin activity 1 (RCAN1): Anti-inflammatory mechanism of PDTC through RCAN1 induction.

Pyrrolidine dithiocarbamate (PDTC) is a thiol compound that elicits anti-inflammatory effects by inhibiting NF-κB signaling. In this study, we report that regulator of calcineurin activity 1 (RCAN1) expression is induced by PDTC treatment and that increased RCAN1 expression is dependent on the generation of reactive oxygen species (ROS) and activation of p38 MAPK and JNK signaling. We also report that the ability of PDTC to induce RCAN1 is mediated by activator protein-1 (AP-1)-dependent gene transcription, and identified a functional AP-1 binding site in the RCAN1 promoter by producing mutations and conducting chromatin immunoprecipitation (ChIP) analyses. Moreover, we show that the PDTC-mediated inhibitory effect on NF-κB signaling is significantly perturbed by knocking out RCAN1. Our data provide the first evidence that PDTC prevents in vivo expression of pro-inflammatory cytokines by inducing RCAN1 expression.

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells.

Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H2O2 accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.

Carmustine induces ERK- and JNK-dependent cell death of neuronally-differentiated PC12 cells via generation of reactive oxygen species.

Accumulation of reactive oxygen species (ROS) caused by the inhibition of glutathione reductase (GR) has been proposed as one of the mechanisms responsible for carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU)-induced cytotoxicity. Since mitogen-activated protein kinases (MAPKs) are known to mediate ROS-dependent cell death in multiple cell types, we examined whether redox-sensitive MAPK activation mediated the carmustine-induced cell death of neuronally differentiated PC12 cells. Carmustine induced a concentration- and time-dependent cell death, which was associated with increased caspase-3 activation, a reduction in GR activity accompanied by a concomitant decrease in reduced glutathione levels, and accumulation of ROS. Carmustine-induced caspase-3 activation and cell death were prevented by pretreatment with anti-oxidants or a reducing agent, indicating that carmustine-induced caspase-3 activation and cell death occur via redox-dependent processes. Carmustine induced phosphorylation of the MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. The activation of these kinases was inhibited by pretreatment with N-acetyl-L-cysteine (NAC). Although all the MAPKs were activated by carmustine, only the inhibitors of JNK and ERK prevented carmustine-induced cell death and caspase-3 activation. Our data suggest that carmustine-induced neurotoxicity is, at least in part, due to the activation of ROS-dependent JNK and ERK signaling.

Activation of adenylate cyclase by forskolin increases the protein stability of RCAN1 (DSCR1 or Adapt78).

Overexpression of Regulator of Calcineurin 1 (RCAN1/DSCR1/Adapt78) is known to inhibit the calcineurin-NFAT dependent signaling pathway. In this report, we find that activation of adenylate cyclase by forskolin increases the expression of RCAN1 through the increase of the protein's half-life. The ability of forskolin to increase the accumulation of RCAN1 protein is significantly inhibited with protein kinase A inhibitors such as KT5720 and H-89. Furthermore, forskolin targets the central and C-terminal region of RCAN1 and enhances the inhibitory effect of RCAN1 on the calcineurin-mediated activation of NFAT. Our findings provide the first evidence that the accumulation of the RCAN1 protein by cAMP acts as an important regulatory mechanism in the control of the calcineurin-dependent cellular pathway.