Dennis A. Ricupero

Mechanisms Regulating the Expression, Self-Maintenance, and Signaling-Function of the Bradykinin B2 and B1 Receptors

Bradykinin (BK) is a potent short‐lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G‐protein‐coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Gαi and Gαq G‐protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg10‐kallidin or desArg9‐BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin‐1 or tumor necrosis factor‐α. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and self‐maintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self‐maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews (Regoli and Barabé, 1980 , Pharmacol Rev 32:1–46; Regoli et al., 1990 , J Cardiovasc Pharmacol 15(Suppl 6):S30–S38; Regoli et al., 1993 , Can J Physiol Pharmacol 71:556–557; Marceau, 1995 , Immunopharmacology 30:1–26; Regoli et al., 1998 , Eur J Pharmacol 348:1–10). J. Cell. Physiol. 193: 275–286, 2002.

Regulation of connective tissue growth factor expression by prostaglandin E2

Transforming growth factor-beta (TGF-beta) stimulates alpha(1)(I) collagen mRNA synthesis in human lung fibroblasts through a mechanism that is partially sensitive to cycloheximide and that may involve synthesis of connective tissue growth factor (CTGF). Northern blot analyses indicate that TGF-beta stimulates time- and dose-dependent increases in CTGF mRNA. In TGF-beta-stimulated fibroblasts, maximal levels of CTGF mRNA (3.7-fold above baseline) occur at 6 h. The TGF-beta-stimulated increase in CTGF mRNA was not blocked by cycloheximide. Nuclear run-on analysis indicates that TGF-beta increases the CTGF transcription rate. The TGF-beta-stimulated increases in CTGF transcription and steady-state levels of CTGF mRNA are attenuated in prostaglandin E(2) (PGE(2))-treated fibroblasts. PGE(2) fails to attenuate luciferase activity induced by TGF-beta in fibroblasts transfected with the TGF-beta-responsive luciferase reporter construct p3TP-LUX. In amino acid-deprived fibroblasts, PGE(2) and insulin regulate alpha(1)(I) collagen mRNA levels without affecting CTGF mRNA levels. The data suggest that the regulation of alpha(1)(I) collagen mRNA levels by TGF-beta and PGE(2) may function through both CTGF-dependent and CTGF-independent mechanisms.

Phenylbutyrate decreases type I collagen production in human lung fibroblasts

Fibrotic lung diseases are characterized by excess extracellular matrix production, in particular type I collagen. Phenylbutyrate (PB) is a non‐toxic pharmacological compound that functions as a weak histone deacetylase inhibitor. In hepatic stellate cells, the synthesis of type I collagen expression is decreased by inhibiting histone acetylation. Our studies examined the regulation of type I collagen by PB in human lung fibroblasts. We found that PB decreases basal and transforming growth factor‐β‐stimulated α1(I) collagen mRNA and protein levels. Northern blot analyses demonstrated that PB decreases steady‐state α1(I) collagen mRNA levels by 78% without significantly changing the stability of the mRNA transcript. PB stimulates cAMP production and increases the acetylation of histone H4, but does not affect the activity of two transforming growth factor‐β (TGF‐β)‐responsive luciferase reporter constructs. These data suggest that PB regulates type I collagen expression in human lung fibroblasts by mechanisms that include cAMP production and histone acetylation. PB may have therapeutic use in fibrotic lung diseases.

INTERLEUKIN-4 REGULATES CONNECTIVE TISSUE GROWTH FACTOR EXPRESSION IN HUMAN LUNG FIBROBLASTS

Transforming growth factor‐β (TGF‐β) and interleukin‐4 (IL‐4) have fibrogenic properties and induce extracellular matrix production in a variety of lung diseases. Connective tissue growth factor (CTGF) is a matrix signaling molecule stimulated by TGF‐β that in part mediates α1(I) collagen mRNA expression. In these studies, the regulation of CTGF expression by IL‐4 in human lung fibroblasts was examined. Following 6 h of stimulation with IL‐4, basal CTGF mRNA levels were unchanged as assessed by Northern blot analysis. However, IL‐4 attenuated the TGF‐β‐stimulated induction of CTGF mRNA expression by 50%. This effect was selective because IL‐4 did not affect fibronectin or α1(I) collagen mRNA expression induced by TGF‐β. Experiments employing the transcriptional inhibitor actinomycin D suggest that IL‐4 did not affect the stability of the CTGF mRNA. Transient transfection assays with 3TP‐Lux, a luciferase gene controlled by a TGF‐β inducible promoter, and with a CTGF promoter construct indicate that IL‐4 interfered with the TGF‐β‐induced transcriptional activation of the CTGF gene. J. Cell. Biochem. 85: 496–504, 2002.

Apigenin decreases expression of the myofibroblast phenotype

We investigated the effect of the dietary flavonoid apigenin on myofibroblast function. We report that in myofibroblasts treated with apigenin, proliferation and basal levels of α1(I) collagen and α‐smooth muscle actin mRNAs were markedly reduced. Apigenin also attenuated the transforming growth factor‐β‐stimulated increases of α1(I) collagen and α‐smooth muscle actin mRNAs. Characterization of the apigenin effects indicates that apigenin reduces both the stability of the α1(I) collagen mRNA and the rate of transcription of the α1(I) collagen gene through a cycloheximide‐sensitive pathway. Western blot analyses indicate that Akt activity is reduced in apigenin‐treated myofibroblasts.

Interactions of bradykinin, calcium, G-protein and protein kinase in the activation of phospholipase A2 in bovine pulmonary artery endothelial cells.

Rise in free cytosolic calcium concentrations [Ca2+]i in response to bradykinin and guanosine 5′-O-thiotriphosphate (GTPτS) was related to the action of phospholipase A2 (arachidonic acid release). At 900 μM extracellular CaCl2, bradykinin induced a typical Ca2+ movement consisting of an initial [Ca2+]i peak at approximately 400 nM followed by a sustained increase in the steady-state cytosolic Ca2+ level at approximately 290 nM. As the extracellular CaCl2 concentration was reduced to 100 μM, the bradykinin induced initial spike was reduced followed by only a marginal increase in steady-state cytosolic Ca2+ levels. Treatment of endothelial cells with saponin (0.002% w/w) did not increase [Ca2+]i and saponin treated cells exhibited a very similar pattern of Ca2+ mobilization in response to bradykinin. However, with saponin treatment, GTPτS (100 μM) increased [Ca2+]i at an almost identical tracing exhibited with 50 nM bradykinin stimulation (in either the presence or absence of 0.002% saponin). No additive increase in [Ca2+]i was observed in cells stimulated with both 100 μM GTPτS and 50 nM bradykinin or in bradykinin stimulated cells subsequently exposed to GTPτS. Pertussis toxin (PTX) did not affect the bradykinin induced Ca2+ mobilization. However, as we showed previously [1], PTX inhibited bradykinin stimulated arachidonic acid release. These results indicate transduction of the bradykinin signal by G-protein for both phospholipase A2 (PLA2) activation and Ca2+ mobilization but likely by different Gα subunits, a PTX sensitive and an insensitive subunit. Furthermore, the bradykinin and GTPτS stimulated release of arachidonic acid appears to be only partially dependent on [Ca2+]i. For example, 10 μM ionomycin, a calcium ionophore, did not release arachidonic acid at extracellular CaCl2 concentrations below 300 μM while GTPτS stimulated a greater release of arachidonic acid at 300 and 100 μM CaCl2 than at 900 μM CaCl2. However, at 100 μM CaCl2, ionomycin increased [Ca2+]i to the same level as bradykinin or GTPτS stimulated cells incubated in 900 μM CaCl2.In previously published experiments [1], we showed that phorbol 12-myristate 13-acetate (TPA) augments bradykinin activated arachidonic acid release in endothelial cells. In the absence of bradykinin, TPA had little effect on arachidonic acid release by endothelial cells. However, in the saponin treated cells, TPA alone (in the absence of bradykinin) caused a marked release of arachidonic acid. The bradykinin and TPA activated arachidonic acid releases were additive. The TPA activated release did not require an increase in [Ca2+]i and occurred in the absence of any added extracellular CaCl2. TPA did not induce an increase in [Ca2+]i in either saponin treated or untreated endothelial cells. This TPA stimulated release of arachidonic acid was totally down-regulated by an 18 h preincubation of the cells in 500 nM TPA but was not inhibited by protein kinase C inhibitor H7.

Na+/Ca2+ Exchanger Activity Modulates Connective Tissue Growth Factor mRNA Expression in Transforming Growth Factor β1- and Des-Arg10-kallidin-stimulated Myofibroblasts

Transforming growth factor (TGF)-β and des-Arg10-kallidin stimulate the expression of connective tissue growth factor (CTGF), a matrix signaling molecule that is frequently overexpressed in fibrotic disorders. Because the early signal transduction events regulating CTGF expression are unclear, we investigated the role of Ca2+ homeostasis in CTGF mRNA expression in TGF-β1- and des-Arg10-kallidin-stimulated human lung myofibroblasts. Activation of the kinin B1 receptor with des-Arg10-kallidin stimulated a rise in cytosolic Ca2+ that was extracellular Na+-dependent and extracellular Ca2+-dependent. The des-Arg10-kallidin-stimulated increase of cytosolic Ca2+ was blocked by KB-R7943, a specific inhibitor of Ca2+ entry mode operation of the plasma membrane Na+/Ca2+ exchanger. TGF-β1 similarly stimulated a KB-R7943-sensitive increase of cytosolic Ca2+ with kinetics distinct from the des-Arg10-kallidin-stimulated Ca2+ response. We also found that KB-R7943 or 2′,4′-dichlorobenzamil, an amiloride analog that inhibits the Na+/Ca2+ exchanger activity, blocked the TGF-β1- and des-Arg10-kallidin-stimulated increases of CTGF mRNA. Pretreatment with KB-R7943 also reduced the basal and TGF-β1-stimulated levels of α1(I) collagen and α smooth muscle actin mRNAs. These data suggest that, in addition to regulating ion homeostasis, Na+/Ca2+ exchanger acts as a signal transducer regulating CTGF, α1(I) collagen, and α smooth muscle actin expression. Consistent with a more widespread role for Na+/Ca2+ exchanger in fibrogenesis, we also observed that KB-R7943 likewise blocked TGF-β1-stimulated levels of CTGF mRNA in human microvascular endothelial and human osteoblast-like cells. We conclude that Ca2+ entry mode operation of the Na+/Ca2+ exchanger is required for des-Arg10-kallidin- and TGF-β1-stimulated fibrogenesis and participates in the maintenance of the myofibroblast phenotype.

Functional expression of the bradykinin-B2 receptor cDNA in Chinese hamster lung CCL39 fibroblasts

The bradykinin (BK) B2 receptor cDNA was synthesized by rt-PCR and transfected into the Chinese hamster lung fibroblasts, CCL39. The CCL39 do not contain the mRNA for this receptor and do not bind BK. Clones of transfected cells were screened for BK receptor mRNA, binding of BK, and for [Ca2+]i response to BK. The clones showed various levels of receptor mRNA. Scatchard analysis of three clones, B6, B5 and B1, each gave a Kd of approximately 1.0nM while the Bmax for each clone differed at 320, 38.7, and 5.39 fmoles per 10(6) cells respectively. The [Ca2+]i response of the three clones to BK decreased with the receptor number/cell. Thus, levels of mRNA, BK binding and [Ca2+]i response proved proportionally related in the transfected clones. The actions of BK and alpha-thrombin, which has an endogenous receptor in these cells, were assessed in clone B6. BK proved active but also distinct from thrombin. BK at 10nM and thrombin at 2units/ml both effectively increased cytosolic [Ca2+]i. BK at 10nM stimulated PGE2 production three fold over basal, while thrombin only marginally elevated PGE2 levels. Alone, BK stimulated a small increase in 3H-thymidine incorporation into DNA. However, in combination with insulin, BK stimulated DNA synthesis to 76% of thrombin, a potent mitogen in these cells. These results illustrate that the BK-B2 receptor cDNA can be stably transfected into a mammalian cell and can activate transmembrane signalling pathways.

Mechanisms in Bradykinin Stimulated Arachidonate Release and Synthesis of Prostaglandin and Platelet Activating Factor

Regulatory mechanisms in bradykinin (BK) activated release of arachidonate (ARA) and synthesis of prostaglandin (PG) and platelet activating factor (PAF) were studied in bovine pulmonary artery endothelial cells (BPAEC). A role for GTP binding protein (G-protein) in the binding of BK to the cells was determined. Guanosine 5-O- (thiotriphosphate), (GTPτS), lowered the binding affinity for BK and increased the Kd for the binding from 0.45 to 1.99 nM. The Bmax remained unaltered at 2.25 × 10-11 mole. Exposure of the cells to aluminium fluoride also reduced the affinity for BK. Bradykinin-induced release of ARA proved pertussis toxin (PTX) sensitive, with a maximum sensitivity at 10 ug/ml PTX. GTPτS at 100 μM increased the release of arachidonate. The effect of GTPτS and BK was additive at suboptimal doses of BK up to 0.5 nM but never exceeded the levels of maximal BK stimulation at 50 nM. PTX also inhibited the release of ARA induced by the calcium ionophore, A23187. Phorbol 12-myristate 13-acetate or more commonly known as tetradecanoyl phorbol acetate (TPA) itself had little effect on release by the intact cells. However, at 100 nM it augmented the BK activated release. This was downregulated by overnight exposure to TPA and correlated with down-regulation of protein kinase C (PKC) activity. The down-regulation only affected the augmentation of ARA release by TPA but not the original BK activated release. TPA displayed a similar, but more potent amplification of PAF synthesis in response to both BK or the calcium ionophore A23187. These results taken together point to the participation of G-protein in the binding of BK to BPAEC and its activation of ARA release. Possibly two types of G-protein are involved, one associated with the receptor, the other activated by Ca2+ and perhaps associated with phospholipase A2 (PLA2). Our results further suggest that a separate route of activation, probably also PLA2 related, takes place through a PKC catalysed phosphorylation.

Amino acid availability regulates type I procollagen accumulation in human lung fibroblasts

Fibrotic lung diseases are characterized by excessive deposition of type I collagen. Amino acid availability regulates type I collagen mRNA levels in quiescent human lung fibroblasts. In these studies, the effect of amino acid availability on type I collagen protein accumulation in quiescent human lung fibroblasts was examined. Following amino acid deprivation, α1(I) procollagen protein levels were not detected by Western blot analysis in either the intracellular or the extracellular compartments. Fibronectin levels and total protein levels were not affected. Amino acid deprivation resulted in a more pronounced decrease in α1(I) procollagen protein levels than in α1(I) procollagen mRNA levels, suggesting that post‐transcriptional events were responsible for the further decrease inα1(I) procollagen protein levels. The addition of transforming growth factor‐β to amino acid deprived fibroblasts increased α1(I) procollagen mRNA levels without affecting α1(I) procollagen protein levels, confirming a post‐transcriptional site for regulatory control by amino acid deprivation. In the absence of ascorbic acid, α1(I) procollagen protein levels increased in amino acid deprived fibroblasts, but α1(I) procollagen mRNA levels were not affected. The absence of ascorbic acid likely resulted in the accumulation of nonhelical procollagen in the endoplasmic reticulum, indicating that translational mechanisms for α1(I) procollagen were intact. The addition of chloroquine, an inhibitor of lysosomal degradation of proteins, increased α1(I) procollagen protein levels in amino acid deprived fibroblasts. These data suggest that following amino acid deprivation of quiescent fibroblasts, newly synthesized type I collagen was degraded intracellularly, primarily by a process that involved lysosomal proteinases. J. Cell. Biochem. 75:130–137, 1999.