Autocrine TGF-alpha is associated with Benzo(a)pyrene-induced mucus production and MUC5AC expression during allergic asthma.

OBJECTS: Benzo(a)pyrene (BaP), an environmental pollutant, is present in high concentrations in urban smog and cigarette smoke and has been reported to promote high mucin 5AC (MUC5AC) expression. Epithelium-derived inflammatory cytokines are considered an important modulator of mucus oversecretion and MUC5AC overexpression. Here, we investigated whether the effect of BaP on MUC5AC overexpression was associated with cytokine autocrine activity in vivo and in vitro. METHODS: In vivo, BALB/c mice were treated with ovalbumin (OVA) in the presence or absence of BaP. Allergy-induced mucus production was assessed by Alcian Blue Periodic acid Schiff (AB-PAS) staining. The human airway epithelial cell line NCI-H292 was used in vitro. MUC5AC and transforming growth factor (TGF)-α mRNA levels were assessed with real-time quantitative PCR. The concentration of cytokines was measured by ELISA. The MUC5AC, p-ERK, ERK, p-EGFR and EGFR proteins were detected by Western blotting in cells or by immunohistochemistry in mouse lungs. Small-interfering RNAs were used for gene silencing. RESULTS: TGF-α was overproduced in the supernatant of NCI-H292 cells treated with BaP. Knockdown of TGF-α expression inhibited the BaP-induced increase in MUC5AC expression and subsequent activation of the EGFR-ERK signalling pathway. Knocking down aryl hydrocarbon receptor (AhR) expression or treatment with an ROS inhibitor (N-acetyl-L-cysteine) could relieve the TGF-α secretion induced by BaP in epithelial cells. In an animal study, coexposure to BaP with OVA increased mucus production, MUC5AC expression and ROS-EGFR-ERK activation in the lung as well as TGF-α levels in bronchoalveolar lavage fluid (BALF). Furthermore, the concentration of TGF-α in BALF was correlated with MUC5AC mRNA levels. Additionally, TGF-α expression was found to be positively correlated with MUC5AC expression in the airway epithelial cells of smokers. Compared with non-smoker asthma patients, TGF-α serum levels were also elevated in smoker asthma patients. CONCLUSION: Autocrine TGF-α was associated with BaP-induced MUC5AC expression in vitro and in vivo. BaP induced TGF-α secretion by activating AhR and producing ROS, which led to activation of the EGFR-ERK pathway.

First description of enhanced expression of transforming growth factor-alpha (TGF-α) and glia maturation factor-beta (GMF-ß) correlate with severity of neuropathology in border disease virus-infected small ruminants.

Border disease (BD) is caused by Pestivirus and characterized by severe neuropathology, and histopathologically observed severe hypomyelination. We have previously shown that small ruminants infected with border disease virus (BDV) play an important role for neuropathology and pathogenesis of severe oxidative damage in brain tissue, neuronal mtDNA; in the production of high pathologic levels of nitric oxide; in glial cell activation and stimulation of intrinsic apoptosis pathway. This study aimed to investigate the relationship between glia maturation factor beta (GMF-ß) and transforming growth factor alpha (TGF-α) expressions and the causes of BDV-induced neuropathology and to investigate their role in neuropathogenesis in a way that was not presented before. Expression levels of GMF-ß and TGF-α were investigated. Results of the study revealed that the levels of GMF-ß (P < 0.005) and TGF-α (P < 0.005) expression in the brain tissue markedly increased in the BDV-infected animals compared to the non-infected healthy control group. While TGF-α expressions were predominantly observed in neurons, GMF-ß expressions were found in astrocytes, glial cells and neurons. These results were reasonable to suggest that BDV-mediated increased GMF-ß might play a pivotal role neuropathogenesis and a different type of role in the mechanism of neurodegeneration/neuropathology in the process of BD. The results also indicated that increased levels of GMF up-regulation in glial cells and neurons causes neuronal destruction, suggesting pathological pathway involving GMF-mediated brain cell cytotoxicity. It is clearly indicated that the cause of astrogliosis is due to severe TGF-a expression. This is the first study to demonstrate the expression of GMF-ß and TGF-α in neurons and reactive glial cells and its association with neuropathology in BD.

Furin-mediated cleavage of Pseudomonas exotoxin-derived chimeric toxins.

Pseudomonas exotoxin (PE) requires proteolytic cleavage to generate a 37-kDa C-terminal fragment that translocates to the cytosol and ADP-ribosylates elongation factor 2. Cleavage within cells is mediated by furin, occurs between arginine 279 and glycine 280, and requires an arginine at both P1 and P4 residues. To study the proteolytic processing of PE-derived chimeric toxins, TGFalpha-PE38 (transforming growth factor fused to the domains II and III of PE) and a mutant form, TGFalpha-PE38gly279, were each produced in Escherichia coli. When assessed on various epidermal growth factor (EGF) receptor-positive cell lines, TGFalpha-PE38 was 100-500-fold more toxic than TGFalpha-PE38gly279. In contrast to PE, where cleavage by furin is only evident at pH 5.5, furin cleaved TGFalpha-PE38 over a broad pH range, while TGFalpha-PE38gly279 was resistant to cleavage. TGFalpha-PE38 was poorly toxic for furin-deficient LoVo cells, unless it was first pretreated in vitro with furin. Furin treatment produced a nicked protein that was 30-fold more toxic than its unnicked counterpart. Using the single chain immunotoxin HB21scFv-PE40 as a substrate, furin-mediated processing of an antibody-based immunotoxin was also evaluated. HB21scFv-PE40, which targets cells expressing the transferrin receptor, was cleaved in a similar fashion to that of TGFalpha-PE38 and nicked HB21scFv-PE40 exhibited increased toxicity for LoVo cells. In short-term experiments, the rate of reduction in protein synthesis by furin-nicked immunotoxins was increased compared with unnicked protein, indicating that cleavage by furin can be a rate-limiting step. We conclude that furin-mediated cleavage of PE-derived immunotoxins is important for their cytotoxic activity.

Small chimeric toxins containing only transforming growth factor alpha and domain III of Pseudomonas exotoxin with good antitumor activity in mice.

Chimeric toxins composed of transforming growth factor alpha (TGF alpha) fused to mutant forms of Pseudomonas exotoxin (PE) bind to the epidermal growth factor receptor and kill cells bearing epidermal growth factor receptors. Initially, the binding domain (Ia; amino acids 1-252) of PE was deleted and replaced with TGF alpha to make TGF alpha-PE40 in which TGF alpha is fused to domains II, Ib, and III of PE (amino acids 253-613). That drug is currently undergoing clinical study for the intravesical therapy of bladder cancer. To generate smaller molecules that would have increased tumor penetration, several deletion mutants were constructed. In one of these, TGF alpha was inserted near the carboxyl terminus of PE, and residues in domains II and Ib of PE (amino acids 253-279 and 365-380) were deleted so that the chimeric toxin did not need to be cleaved by an intracellular protease to be activated (Theuer et al., J. Biol. Chem., 267: 16872-16877, 1992). We have now constructed chimeric toxins which contain only domain III, yet still exhibit high cytotoxic activity on epidermal growth factor receptor-containing cells and produce substantial tumor regressions in mice bearing a human xenograft. The high cytotoxic activity of these severely truncated toxins provides new insights on the proposed functions of domains II and III of PE.

Selective toxicity of TGF-alpha-PE40 to EGFR-positive cell lines: selective protection of low EGFR-expressing cell lines by EGF.

The sensitivity of human breast and lung cancer cell lines to TGF-alpha-PE40, a novel chimeric recombinant cytotoxin composed of two independent domains, (i) TGF-alpha and (ii) a 40 kDa segment of the Pseudomonas exotoxin protein, PE-40, was investigated. Toxicity varied widely, correlated with epidermal growth factor receptor (EGFR) levels (P = 0.01) and was greatly reduced by EGF, indicating that binding of TGF-alpha-PE40 to EGFR is important in mediating toxicity. Cell lines expressing low EGFR levels were most highly protected by EGF, indicating that normal (low EGFR-expressing) tissue may be selectively protected by EGF in vivo. P-glycoprotein did not confer resistance to TGF-alpha-PE40, and toxicity was unaffected by multidrug resistance-modulating agents (cyclosporin A, tamoxifen, verapamil), indicating a role for TGF-alpha-PE40 in the clinical management of drug-resistant tumours.

Transgenic mice provide genetic evidence that transforming growth factor alpha promotes skin tumorigenesis via H-ras-dependent and H-ras-independent pathways.

The epidermal growth factor receptor (EGFR), which mediates the mitogenic activity of transforming growth factor alpha (TGF-alpha), has been shown to activate Ras in cultured cells through well-defined intermediary proteins. To examine the in vivo relationship between EGFR and Ras, chemical carcinogenesis of TGF-alpha transgenic mouse skin was chosen as an experimental model. Transgenic mice overexpressing TGF-alpha in a wide variety of epithelial tissues by virtue of a metallothionein promoter demonstrate a multitude of premalignant and neoplastic lesions but not spontaneous skin tumors. Transgenic skin was initiated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA), shown previously to induce, in concert with a tumor promoter, murine papillomas that consistently contain specific H-ras mutations. Virtually all DMBA-treated TGF-alpha transgenic mice, but not treated control animals, developed hyperplasias, papillomas, sebaceous adenomas, and more infrequently, sebaceous and squamous cell carcinomas. Therefore, TGF-alpha functions as an autonomous tumor promoter in DMBA-initiated transgenic skin. Skin tumors could be separated into two mutually exclusive genetic classes. In tumors harboring mutant H-ras, TGF-alpha transgene expression was relatively low and essentially unchanged relative to untreated skin; however, only 42% of skin tumors contained mutations in H-ras. Conversely, in most tumors with wild-type H-ras, transgenic TGF-alpha transcripts were enhanced 10- to 20-fold. These results suggest that strong constitutive EGFR stimulation, through TGF-alpha transgene overexpression, can substitute functionally for mutational activation of H-ras in skin tumorigenesis. Moreover, because H-ras mutational activation could not induce skin tumors without TGF-alpha transgene activity, simultaneous stimulation of an EGFR-mediated H-Ras-independent pathway appears to be required for tumor development as well.

Transforming growth factor-alpha-Pseudomonas exotoxin fusion protein (TGF-alpha-PE38) treatment of subcutaneous and intracranial human glioma and medulloblastoma xenografts in athymic mice.

Epidermal growth factor receptor (EGFR) is amplified or overexpressed in many malignant gliomas and other primary brain tumors but is low or undetectable in normal brain. In the present study, this differential expression has been exploited for targeted brain tumor therapy using a TGF-alpha-Pseudomonas exotoxin recombinant toxin, TGF-alpha-PE38. In vitro experiments demonstrate that the cytotoxicity of this fusion protein is primarily determined by tumor EGFR expression and that TGF-alpha-PE38 cytotoxicity is abolished by pretreatment with excess epidermal growth factor. Treatment with i.p. TGF-alpha-PE38 in nude mice bearing glioblastoma or medulloblastoma s.c. xenografts produced tumor regression and growth delay. For intracranial xenograft implants treated with i.p. TGF-alpha-PE38, significant increases in median survival were noted only for tumors with the highest EGFR expression. However, intracranial tumors treated with a single intratumoral injection of TGF-alpha-PE38 showed increased survival in all xenografts tested. These results indicate that TGF-alpha-PE38 is active against primary human brain tumors ranging from moderate to high EGFR expression. For intracranial tumors, however, the higher survival rates produced by intracranial injection of TGF-alpha-PE38 than by continuous i.p. administration suggest that increased drug clearance or impaired drug delivery reduces the efficacy of systemic TGF-alpha-PE38. Direct delivery of TGF-alpha-PE38 into brain tumors by controlled-release biodegradable polymers or intratumoral implanted catheters, or intrathecal administration into the colony stimulating factor of patients with leptomeningeal metastasis, may represent clinically useful applications of recombinant toxin therapy in tumors with high EGFR expression.

Rational design of a chimeric toxin: an intramolecular location for the insertion of transforming growth factor alpha within Pseudomonas exotoxin as a targeting ligand.

To investigate the potential utility of Pseudomonas exotoxin (PE) in forming rationally designed chemotherapeutic agents, we inserted a cDNA encoding transforming growth factor alpha (TGF alpha) at several locations in a gene encoding a mutant full-length PE (PE4E) which does not bind to the PE receptor. After expression in Escherichia coli, we purified the chimeric toxins to near homogeneity and showed that they were specifically cytotoxic to human epidermoid, ovarian, colon, and hepatocellular carcinoma lines. Like the previously reported TGF alpha-PE40, one of the new molecules (TGF alpha-PE4E) contains the ligand at the amino terminus. Two additional chimeras (PE4E-TGF alpha and PE4E-TGF alpha-598-613) each contain TGF alpha inserted near the carboxyl terminus of PE. We show that preservation of the correct PE carboxyl-terminal amino acid sequence, REDLK, allows the toxins containing TGF alpha carboxyl inserts to retain significant cytotoxicity against target cells, since another molecule (PE4E-TGF alpha-ILK) containing a nonfunctional carboxyl-terminal sequence was over 100-fold less active. The chimeric toxins with TGF alpha had the same binding affinity for the EGF receptor whether the ligand occupied the amino or carboxyl position. Molecules with TGF alpha near the carboxyl position were consistently less active against target cells but also less toxic to mice than those with TGF alpha at the amino terminus, indicating both types of molecules might be therapeutically effective. Our results establish that a ligand can be placed near the carboxyl terminus of PE, within the portion of the toxin that translocates to the cytosol. The amino-terminal position in such molecules is then available for the placement of other targeting ligands.

Properties of chimeric toxins with two recognition domains: interleukin 6 and transforming growth factor alpha at different locations in Pseudomonas exotoxin.

Pseudomonas exotoxin (PE) is a potent cytotoxic agent that is composed of 613 amino acids arranged into three major domains. We have previously identified two positions where ligands can successfully be placed in PE to direct it to cells with specific surface receptors. One site is at the amino terminus and the other is close to but not at the C-terminus. To examine the possibility of constructing oncotoxins with two different recognition elements that will bind to two different receptors, we have placed cDNAs encoding either transforming growth factor alpha (TGF alpha) or interleukin 6 (IL6) at the 5' end of a PE gene and also inserted a cDNA encoding TGF alpha near the 3' end of the PE gene. The plasmids encoding these chimeric toxins were expressed in Escherichia coli and the chimeric proteins purified to near homogeneity. In all the new toxins, the TGF alpha near the C-terminus was inserted after amino acid 607 of PE and followed by amino acids 604-613 so that the correct PE C-terminus (REDLK) was preserved. For each chimera, the toxin portion was either PE4E, in which the cell binding domain (domain Ia) is mutated, PE40, in which domain Ia is deleted, or PE38, in which domain Ia and part of domain Ib are deleted. These derivatives of PE do not bind to the PE receptor and allow 607, 355, or 339 amino acids, respectively, between the two ligands.(ABSTRACT TRUNCATED AT 250 WORDS)