Cigarette Smoke Regulates the Competitive Interactions between NRF2 and BACH1 for Heme Oxygenase-1 Induction.

Cigarette smoke has been shown to trigger aberrant signaling pathways and pathophysiological processes; however, the regulatory mechanisms underlying smoke-induced gene expression remain to be established. Herein, we observed that two smoke-responsive genes, HO-1 and CYP1A1, are robustly induced upon smoke by different mechanisms in human bronchial epithelia. CYP1A1 is mediated by aryl hydrocarbon receptor signaling, while induction of HO-1 is regulated by oxidative stress, and suppressed by N-acetylcysteine treatment. In light of a pivotal role of NRF2 and BACH1 in response to oxidative stress and regulation of HO-1, we examined if smoke-induced HO-1 expression is modulated through the NRF2/BACH1 axis. We demonstrated that smoke causes significant nuclear translocation of NRF2, but only a slight decrease in nuclear BACH1. Knockdown of NRF2 attenuated smoke-induced HO-1 expression while down-regulation of BACH1 had stimulatory effects on both basal and smoke-induced HO-1 with trivial influence on NRF2 nuclear translocation. Chromatin immunoprecipitation assays showed that smoke augments promoter-specific DNA binding of NRF2 but suppresses BACH1 binding to the HO-1 promoter ARE sites, two of which at -1.0 kb and -2.6 kb are newly identified. These results suggest that the regulation of NRF2 activator and BACH1 repressor binding to the ARE sites are critical for smoke-mediated HO-1 induction.

Targeting myristoylated alanine-rich C kinase substrate phosphorylation site domain in lung cancer. Mechanisms and therapeutic implications.

RATIONALE: Phosphorylation of myristoylated alanine-rich C kinase substrate (phospho-MARCKS) at the phosphorylation site domain (PSD) is crucial for mucus granule secretion and cell motility, but little is known concerning its function in lung cancer. OBJECTIVES: We aimed to determine if MARCKS PSD activity can serve as a therapeutic target and to elucidate the molecular basis of this potential. METHODS: The clinical relevance of phospho-MARCKS was first confirmed. Next, we used genetic approaches to verify the functionality and molecular mechanism of phospho-MARCKS. Finally, cancer cells were pharmacologically inhibited for MARCKS activity and subjected to functional bioassays. MEASUREMENTS AND MAIN RESULTS: We demonstrated that higher phospho-MARCKS levels were correlated with shorter overall survival of lung cancer patients. Using shRNA silencing and ectopic expression of wild-type and PSD-mutated (S159/163A) MARCKS, we showed that elevated phospho-MARCKS promoted cancer growth and erlotinib resistance. Further studies demonstrated an interaction of phosphoinositide 3-kinase with MARCKS, but not with phospho-MARCKS. Interestingly, phospho-MARCKS acted in parallel with increased phosphatidylinositol (3,4,5)-triphosphate pools and AKT activation in cells. Through treatment with a 25-mer peptide targeting the MARCKS PSD motif (MPS peptide), we were able to suppress tumor growth and metastasis in vivo, and reduced levels of phospho-MARCKS, phosphatidylinositol (3,4,5)-triphosphate, and AKT activity. This peptide also enhanced the sensitivity of lung cancer cells to erlotinib treatment, especially those with sustained activation of phosphoinositide 3-kinase/AKT signaling. CONCLUSIONS: These results suggest a key role for MARCKS PSD in cancer disease and provide a unique strategy for inhibiting the activity of MARCKS PSD as a treatment for lung cancer.

Characterization of a novel long noncoding RNA, SCAL1, induced by cigarette smoke and elevated in lung cancer cell lines.

The incidence of lung diseases and cancer caused by cigarette smoke is increasing. The molecular mechanisms of gene regulation induced by cigarette smoke that ultimately lead to cancer remain unclear. This report describes a novel long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) both in vitro and in vivo and is elevated in numerous lung cancer cell lines. We have termed this lncRNA the smoke and cancer-associated lncRNA-1 (SCAL1). This lncRNA is located in chromosome 5, and initial sequencing analysis reveals a transcript with four exons and three introns. The expression of SCAL1 is regulated transcriptionally by nuclear factor erythroid 2-related factor (NRF2), as determined by the small, interfering RNA (siRNA) knockdown of NRF2 and kelch-like ECH-associated protein 1 (KEAP1). A nuclear factor erythroid-derived 2 (NF-E2) motif was identified in the promoter region that shows binding to NRF2 after its activation. Functionally, the siRNA knockdown of SCAL1 in human bronchial epithelial cells shows a significant potentiation of cytotoxicity induced by CSE in vitro. Altogether, these results identify a novel and intriguing new noncoding RNA that may act downstream of NRF2 to regulate gene expression and mediate oxidative stress protection in airway epithelial cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced MUC5AC expression: aryl hydrocarbon receptor-independent/EGFR/ERK/p38-dependent SP1-based transcription.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxicant. Epidemiological studies have associated TCDD exposure with the development of chronic obstructive pulmonary disease, which is manifested by mucous/goblet cell hyperplasia. The purpose of this research was to elucidate the pathway/mechanisms that lead to TCDD-induced gene expression in both primary normal human bronchial epithelial cells and an immortalized cell line, HBE1, under air-liquid interface conditions. TCDD exposure induced a time-dependent elevation of MUC5AC mRNA and protein synthesis, and cytochrome p450 1A1 (CYP1A1) expression in these cells. Treatment with an aryl hydrocarbon receptor antagonist had no effect on TCDD-induced MUC5AC expression, but significantly suppressed CYP1A1 induction. However, treatments with inhibitors of signaling pathways and the expression of dominant negative mutants of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) and p38, but not the inhibition of c-Jun N-terminal kinase pathway, abrogated MUC5AC induction, but not that of CYP1A1. These effects also occurred at the MUC5AC promoter-reporter level using the chimeric construct for a transient transfection study. Western blot analysis confirmed the phosphorylation of activated EGFR, ERK, and p38 signaling molecules, but not the c-Jun N-terminal kinase, in cells after TCDD exposure. Specificity protein 1 (Sp1) phosphorylation also occurred in cells after TCDD exposure. Both MUC5AC expression and the promoter activity were inhibited by mithramycin A, an inhibitor specific to Sp1-based transcription. These results lead to the conclusion that TCDD induced MUC5AC expression through a noncanonical aryl hydrocarbon receptor-independent, EGFR/ERK/p38-mediated signaling pathway-mediated/Sp1-based transcriptional mechanism.

NF-κB mediates IL-1ß- and IL-17A-induced MUC5B expression in airway epithelial cells.

A major pathological feature of chronic airway diseases is the elevated expression of gel-forming mucins. NF-κB activation in airway epithelial cells has been shown to play a proinflammatory role in chronic airway diseases; however, the specific role of NF-κB in mucin gene expression has not been characterized. In this study, we show that the proinflammatory cytokines, IL-1ß and IL-17A, both of which use the NF-κB pathway, are potent inducers of MUC5B mRNA expression in both well differentiated primary normal human bronchial epithelial cells and the human bronchial epithelial cell line, HBE1. MUC5B induction by these cytokines was both time- and dose-dependent, and was attenuated by the small molecule inhibitor, NF-κB inhibitor III, as well as p65 small interfering RNA, suggesting that the regulation of MUC5B expression by these cytokines is via an NF-κB-based transcriptional mechanism. Deletion analysis of the MUC5B promoter demonstrated that IL-1ß- and IL-17A-induced promoter activity resides within the -4.17-kb to -2.56-kb region relative to the transcriptional start site. This region contains three putative κB-binding sites (NF-κB-1, -3,786/-3,774; NF-κB-2, -3,173/-3,161; and NF-κB-3, -2,921/-2,909). Chromatin immunoprecipitation analysis confirmed enhanced binding of the p50 NF-κB subunit to the NF-κB-3 site after cytokine stimulation. We conclude that an NF-κB-based transcriptional mechanism is involved in MUC5B regulation by IL-1ß and IL-17A in airway epithelium. This is the first demonstration of the participation of NF-κB and its specific binding site in cytokine-mediated airway MUC5B expression.

Regulation of airway MUC5AC expression by IL-1beta and IL-17A; the NF-kappaB paradigm.

Mucin over-production is one of the hallmarks of chronic airway diseases such as chronic obstructive pulmonary disease, asthma, and cystic fibrosis. NF-kappaB activation in airway epithelial cells has been shown to play a positive inflammatory role in chronic airway diseases; however, the role of NF-kappaB in mucin gene expression is unresolved. In this study, we have shown that the proinflammatory cytokines, IL-1beta and IL-17A, both of which utilize the NF-kappaB pathway, are potent inducers of mucin (MUC)5AC mRNA and protein synthesis by both well-differentiated primary normal human bronchial epithelial cells and the human bronchial epithelial cell line, HBE1. MUC5AC induction by these cytokines was both time- and dose-dependent and occurred at the level of promoter activation, as measured by a reporter gene assay. These effects were attenuated by the small molecule inhibitor NF-kappaB inhibitor III, as well as p65 small-interfering RNA, suggesting that the regulation of MUC5AC expression by these cytokines is via an NF-kappaB-based transcriptional mechanism. Further investigation of the promoter region identified a putative NF-kappaB binding site at position-3594/-3582 in the promoter of MUC5AC as critical for the regulation of MUC5AC expression by both IL-1beta and IL-17A. Chromatin immunoprecipitation analysis confirmed enhanced binding of the NF-kappaB subunit p50 to this region following cytokine stimulation. We conclude that an NF-kappaB-based transcriptional mechanism is involved in MUC5AC regulation by IL-1beta and IL-17A in the airway epithelium. This is the first demonstration of the participation of NF-kappaB and its specific binding site in cytokine-mediated airway MUC5AC expression.

Potentiation of IL-19 expression in airway epithelia by IL-17A and IL-4/IL-13: important implications in asthma.

BACKGROUND: IL-17A and IL-19 are highly expressed in chronic inflammatory diseases, such as psoriasis and asthma. IL-19 plays a significant role in the enhancement of T(H)2 cytokine secretion in allergic diseases, but its cellular source in asthmatic patients remains unknown. OBJECTIVE: Our aims were to determine whether the epithelium is a major source of airway mucosal IL-19 and to elucidate the mechanism of gene expression regulation. METHODS: Immunofluorescent staining was used to determine IL-19 protein expression in tracheal tissue sections of various airway diseases. Well-differentiated primary human bronchial epithelial cultures and a corresponding cell line were used as in vitro models to study gene regulation. RESULTS: We found significantly higher IL-19 expression in airway epithelia of asthmatic patients than in epithelia of patients with other diseases. Using a cytokine panel, we demonstrated the upregulation of IL-19 expression in cultures by two T(H)2 cytokines, IL-4 and IL-13, in addition to the previously found T(H)17 cytokine IL-17A. Moreover, cotreatment of IL-17A and IL-4/IL-13 synergistically upregulated IL-19 expression. Using siRNA and chemical inhibitor approaches, we demonstrated a transcriptional regulation of IL-19 by nuclear factor kappaB and signal transducer and activator of transcription (STAT) 6. The addition of IL-13 to IL-17A stimulation triggers a shift from nuclear factor kappaB-dependent transcriptional regulation to one that is STAT6 based. Using chromatin immunoprecipitation assays, we demonstrated the presence of STAT6-binding elements in the IL-19 promoter region. CONCLUSION: We propose that an IL-17A- and IL-13-induced synergism in IL-19 stimulation in airway epithelia occurs through a STAT6-dependent pathway.

Regulation of airway mucin gene expression.

Mucins are important components that exert a variety of functions in cell-cell interaction, epidermal growth factor receptor signaling, and airways protection. In the conducting airways of the lungs, mucins are the major contributor to the viscoelastic property of mucous secretion, which is the major barrier to trapping inhaled microbial organism, particulates, and oxidative pollutants. The homeostasis of mucin production is an important feature in conducting airways for the maintenance of mucociliary function. Aberrant mucin secretion and accumulation in airway lumen are clinical hallmarks associated with various lung diseases, such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, and lung cancer. Among 20 known mucin genes identified, 11 of them have been verified at either the mRNA and/or protein level in airways. The regulation of mucin genes is complicated, as are the mediators and signaling pathways. This review summarizes the current view on the mediators, the signaling pathways, and the transcriptional units that are involved in the regulation of airway mucin gene expression. In addition, we also point out essential features of epigenetic mechanisms for the regulation of these genes.

Regulation of airway innate and adaptive immune responses: the IL-17 paradigm.

IL-17 is a proinflammatory cytokine produced by immune cells. Its significance in host defense and disease development has been demonstrated in various infection and autoimmune models. Recently, additional studies have shown that IL-17 is also important in modulating airway immune response in several aspects. Along with the well-established Th1/Th2 model, new discoveries regarding the Th17 lineage and IL-17 functions have added an extra twist to the already complicated cytokine network that regulates airway immunity. The IL-17 receptor is expressed on blood cells, as well as on structural cells such as the epithelial cells in the airway. Therefore, the effect of IL-17 on airway immunity is very broad, covering both the innate and the adaptive aspects. In this review, we summarize the findings of recent studies on IL-17 signaling and function in pulmonary immune response, and the implications of IL-17 in disease pathogenesis.

Requirement for both JAK-mediated PI3K signaling and ACT1/TRAF6/TAK1-dependent NF-kappaB activation by IL-17A in enhancing cytokine expression in human airway epithelial cells.

Through DNA microarray analysis and quantitative PCR verification, we have identified additional IL-17A-inducible genes-IL-19, CXCL-1, -2, -3, -5, and -6-in well-differentiated normal human bronchial epithelial cells. These genes, similar to previously described human beta-defensin-2 (HBD-2) and CCL-20, were induced by a basolateral treatment of IL-17A, and regulated by PI3K signaling and NF-kappaB activation. For PI3K signaling, increases of cellular PIP(3) and phosphorylation of downstream molecules, such as Akt and glycogen synthase kinase-3beta (GSK3beta) (S9), were detected. Induced gene expression and HBD-2 promoter activity were attenuated by LY294002, p110alpha small-interfering RNA (siRNA), as well as by an overexpression of constitutively active GSK3beta(S9A) or wild-type phosphatase and tensin homolog. Increased phosphorylation of JAK1/2 after IL-17A treatment was detected in primary normal human bronchial epithelium cells. Transfected siRNAs of JAK molecules and JAK inhibitor I decreased IL-17A-induced gene expression and GSK3beta(S9) phosphorylation. However, both JAK inhibitor I and PI3K inhibitor had no effect on the DNA-binding activities of p65 and p50 to NF-kappaB consensus sequences. This result suggested a JAK-associated PI3K signaling axis is independent from NF-kappaB activation. With siRNA to knockdown STIR (similar expression to fibroblast growth factor and IL-17R; Toll-IL-1R)-related signaling molecules, such as Act1, TNFR-associated factor 6 (TRAF6), and TGF-beta-activated kinase 1 (TAK1), and transfection of A52R, an inhibitor of the MyD88/TRAF6 complex, or dominant-negative TAK1, IL-17A-inducible gene expression and HBD-2 promoter activity were reduced. Additionally, IL-17A-induced p65 and p50 NF-kappaB activations were confirmed and their nuclear translocations were down-regulated by siRNAs of TRAF6 and TAK1. These results suggest that two independent and indispensable signaling pathways-1) JAK1-associated PI3K signaling and 2) Act1/TRAF6/TAK1-mediated NF-kappaB activation-are stimulated by IL-17A to regulate gene induction in human airway epithelial cells.

Up-regulation of CC chemokine ligand 20 expression in human airway epithelium by IL-17 through a JAK-independent but MEK/NF-kappaB-dependent signaling pathway.

CCL20, like human beta-defensin (hBD)-2, is a potent chemoattractant for CCR6-positive immature dendritic cells and T cells in addition to recently found antimicrobial activities. We previously demonstrated that IL-17 is the most potent cytokine to induce an apical secretion and expression of hBD-2 by human airway epithelial cells, and the induction is JAK/NF-kappaB-dependent. Similar to hBD-2, IL-17 also induced CCL20 expression, but the nature of the induction has not been elucidated. Compared with a panel of cytokines (IL-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, IFN-gamma, GM-CSF, and TNF-alpha), IL-17 was as potent as IL-1alpha, 1beta, and TNF-alpha, with a time- and dose-dependent phenomenon in stimulating CCL20 expression in both well-differentiated primary human and mouse airway epithelial cell culture systems. The stimulation was largely dependent on the treatment of polarized epithelial cultures from the basolateral side with IL-17, achieving an estimated 4- to 10-fold stimulation at both message and protein levels. More than 90% of induced CCL20 secretion was toward the basolateral compartment (23.02 +/- 1.11 ng/chamber/day/basolateral vs 1.82 +/- 0.82 ng/chamber/day/apical). Actinomycin D experiments revealed that enhanced expression did not occur at mRNA stability. Inhibitor studies showed that enhanced expression was insensitive to inhibitors of JAK/STAT, p38, JNK, and PI3K signaling pathways, but sensitive to inhibitors of MEK1/2 and NF-kappaB activation, suggesting a MEK/NF-kappaB-based mechanism. These results suggest that IL-17 can coordinately up-regulate both hBD-2 and CCL20 expressions in airways through differentially JAK-dependent and -independent activations of NF-kappaB-based transcriptional mechanisms, respectively.

Differential regulation of MUC5AC/Muc5ac and hCLCA-1/mGob-5 expression in airway epithelium.

This study demonstrates that the two biomarkers, MUC5AC/ Muc5ac and hCLCA1/Gob5, which are frequently associated with surface mucous/goblet cells in asthmatic airways, are differentially regulated. Intratracheal instillation of IL-13 (0.5 mug/mouse lung) elicited 8- and 110-fold induction of Muc5ac and Gob5 messages, respectively, within 24 h in wild-type mouse lung, whereas these inductions were abrogated in Stat6 knockout mice. The induction of MUC5AC/Muc5ac message could not be duplicated in vitro with primary tracheobronchial epithelial (TBE) cells derived from wild-type mice or humans, despite significant inductions still seen for hCLCA1/Gob5. Further studies with JAK inhibitors and STAT6 signaling showed active signaling of the JAK/STAT6 pathway in these primary TBE cultures by IL-13 in the regulation of hCLCA1 expression. Dual immunofluorescent staining with antibodies specific to MUC5AC and hCLCA1 revealed a differential nature of the expression of these two biomarkers by distinct cell types of primary TBE cultures. Finally, MUC5AC expression could be elevated by a bacterial product, peptidoglycan, without any induction of hCLCA1. Thus, these results suggest that the two biomakers of the metaplastic airway mucous cell type are differentially regulated by JAK/STAT6-dependent and -independent pathways.

Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium.

Partially reduced metabolites of molecular oxygen, superoxide (O2-) and hydrogen peroxide (H2O2), are detected in respiratory tract lining fluid, and it is assumed that these are key components of innate immunity. Whether these reactive oxygen species (ROS) are produced specifically by the respiratory epithelium in response to infection, or are a non-specific by-product of oxidant-producing inflammatory cells is not well characterized. Increasing evidence supports the hypothesis that the dual function NAD(P)H oxidases/peroxidases, Duox1 and Duox2, are important sources of regulated H2O2 production in respiratory tract epithelium. However, no studies to date have characterized the regulation of Duox gene expression. Accordingly, we examined Duox1 and Duox2 mRNA expression by real-time PCR in primary respiratory tract epithelial cultures after treatment with multiple cytokines. Herein, we determined that Duox1 expression was increased several-fold by treatment with the Th2 cytokines IL-4 and IL-13, whereas Duox2 expression was highly induced following treatment with the Th1 cytokine IFN-gamma. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H2O2 production was similarly increased by the addition of Th1 or Th2 cytokines. These results demonstrate for the first time the regulation of Duox expression by immunomodulatory Th1 and Th2 cytokines, and suggest a mechanism by which ROS production can be regulated in the respiratory tract as part of the host defense response.

IL-17 markedly up-regulates beta-defensin-2 expression in human airway epithelium via JAK and NF-kappaB signaling pathways.

Using microarray gene expression analysis, we first observed a profound elevation of human beta-defensin-2 (hBD-2) message in IL-17-treated primary human airway epithelial cells. Further comparison of this stimulation with a panel of cytokines (IL-1alpha, 1beta, 2-13, and 15-18; IFN-gamma; GM-CSF; and TNF-alpha) demonstrated that IL-17 was the most potent cytokine to induce hBD-2 message (>75-fold). IL-17-induced stimulation of hBD-2 was time and dose dependent, and this stimulation also occurred at the protein level. Further studies demonstrated that hBD-2 stimulation was attenuated by IL-17R-specific Ab, but not by IL-1R antagonist or the neutralizing anti-IL-6 Ab. This suggests an IL-17R-mediated signaling pathway rather than an IL-17-induced IL-1alphabeta and/or IL-6 autocrine/paracrine loop. hBD-2 stimulation was sensitive to the inhibition of the JAK pathway, and to the inhibitors that affect NF-kappaB translocation and the DNA-binding activity of its p65 NF-kappaB subunit. Transient transfection of airway epithelial cells with an hBD-2 promoter-luciferase reporter gene expression construct demonstrated that IL-17 stimulated promoter-reporter gene activity, suggesting a transcriptional mechanism for hBD-2 induction. These results support an IL-17R-mediated signaling pathway involving JAK and NF-kappaB in the transcriptional stimulation of hBD-2 gene expression in airway epithelium. Because IL-17 has been identified in a number of airway diseases, especially diseases related to microbial infection, these findings provide a new insight into how IL-17 may play an important link between innate and adaptive immunity, thereby combating infection locally within the airway epithelium.

Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop.

Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleukin-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor alpha). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B and MUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5B expression through the ERK signaling pathway.

Branched-chain 6-ketoacid dehydrogenase kinase: A mammalian enzyme with histidine kinase activity.

Whereas phosphoesters of serine, threonine, and tyrosine are present in great abundance in mammalian cells, only limited information is available for other amino acids modified by a phosphate group. Phosphohistidine in proteins has been discovered in mammalian cells, but no enzyme with histidine kinase activity has been reported to date. The present study demonstrates for the first time the histidine kinase activity of a mammalian protein. Branched-chain alpha-ketoacid dehydrogenase kinase, a mitochondrial enzyme, has high sequence similarity with histidine kinases from lower organisms but has been classified as a serine/threonine kinase. Our studies indicate that in addition to a serine this enzyme also autophosphorylates a histidine residue. This finding suggests that histidine kinases are not restricted to lower organisms.