Esophageal pepsin and proton pump synthesis in barrett's esophagus and esophageal adenocarcinoma.

OBJECTIVES/HYPOTHESIS: Gastroesophageal reflux disease and associated metaplasia of the esophagus (Barrett's esophagus [BE]) are primary risk factors for esophageal adenocarcinoma (EAC). Widespread use of acid suppression medications has failed to stem the rise of EAC, suggesting that nonacid reflux may underlie its pathophysiology. Pepsin is a tumor promoter in the larynx and has been implicated in esophageal carcinogenesis. Herein, specimens from the esophageal cancer spectrum were tested for pepsin presence. Pepsin-induced carcinogenic changes were assayed in an esophageal cell culture model. STUDY DESIGN: Laboratory analysis. METHODS: Pepsin was assayed in reflux and cancer free esophagi, BE, EAC, and esophageal cancer lacking association with reflux (squamous cell carcinoma [SCC]). Refluxed or locally synthesized pepsin was assayed by Western blot. Local synthesis of pepsin and proton pumps was assayed via reverse transcription-polymerase chain reaction. The effect of pepsin on BE and EAC markers was investigated via enzyme-linked immunosorbent assay and quantitative polymerase chain reaction in human esophageal epithelial cells treated with pepsin or control diluent. RESULTS: Pepsinogen and proton pump mRNA were observed in BE (3/5) and EAC (4/4) samples, but not in normal adjacent specimens, SCC (0/2), or reflux and cancer-free esophagi. Chronic pepsin treatment (0.1-1 mg/mL, 4 weeks) of human esophageal cells in vitro induced BE and EAC markers interleukin 8 and KRT8 and depleted normal esophageal marker KRT10 (P < .05) expression. CONCLUSIONS: Local synthesis of pepsin and proton pumps in BE and EAC is not uncommon. Absence of these molecules in normal (noncancer) esophagi, SCC, and in vitro data support a role for pepsin in reflux-attributed carcinogenic changes in the esophagus. LEVEL OF EVIDENCE: NA Laryngoscope, 129:2687-2695, 2019.

Is Pepsin a Reliable Marker of Laryngopharyngeal Reflux? A Systematic Review.

Objective Laryngopharyngeal reflux (LPR) is a common illness of otolaryngology visits. Over the past few years, pepsin has become a promising marker of LPR. The objective of the present research is to analyze the existing literature using pepsin as a diagnostic tool of LPR through a systematic review. Data Sources PubMed (Medline), Trip Database, Cochrane Library, EMBASE, SUMsearch, and Web of Science. Review Methods The outcome assessed was the presence of pepsin in LPR patients. We included articles in which pepsin was studied in LPR patients (clinically suspected or with confirmed diagnosis). Studies with no control group, comparison group, and/or a sample size lower than 20 patients were excluded. Results Twelve studies were included. All included studies, with the exception of 2, found statistically significant differences for pepsin in cases compared with healthy controls. Conclusion Pepsin might be a reliable marker in LPR patients, although questions remain about optimal timing, location, nature, and threshold values for pepsin testing. Future investigations are necessary to clarify the best method to use pepsin in the diagnostic process of LPR.

Local Synthesis of Pepsin in Barrett's Esophagus and the Role of Pepsin in Esophageal Adenocarcinoma.

OBJECTIVE: Despite widespread use of proton pump inhibitors (PPIs), the incidence of esophageal adenocarcinoma (EAC) continues to rise. PPIs reduce reflux acidity, but only transiently inactivate gastric enzymes. Nonacid reflux, specifically nonacid pepsin, contributes to carcinogenesis in the larynx. Given the carcinogenic potential of pepsin and inefficacy of PPIs to prevent EAC, the presence and effect of pepsin in the esophagus should be investigated. METHODS: Normal and Barrett's biopsies from 8 Barrett's esophagus patients were collected for pepsin analysis via Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Human esophageal cells cultured from healthy patients were treated with pepsin (0.01-1 mg/mL; 1-20 hours), acid (pH 4)±pepsin (5 minutes); real-time RT-PCR, ELISA, and cell migration were assayed. RESULTS: Pepsin was detected in all 8 Barrett's and 4 of 8 adjacent normal specimens. Pepsinogen mRNA was observed in 22 Barrett's, but not in normal adjacent samples. Pepsin induced PTSG2 (COX-2) and IL-1ß expression and cell migration in vitro. CONCLUSIONS: Pepsin is synthesized by metaplastic, Barrett's esophageal mucosa. Nonacid pepsin increases metrics of tumorigenicity in esophageal epithelial cells in vitro. These findings implicate refluxed and locally synthesized pepsin in development and progression of EAC and, in part, explain the inefficacy of PPIs in the prevention of EAC.

Role of pepsin and pepsinogen: linking laryngopharyngeal reflux with otitis media with effusion in children.

OBJECTIVES/HYPOTHESIS: To analyze the relationship between laryngopharyngeal reflux (LPR) represented by pepsin and pepsinogen, and pathogenesis of otitis media with effusion (OME). STUDY DESIGN: Prospective case-control study. METHODS: Children with OME who required adenoidectomy and tympanostomy/tympanostomy tubes placement were enrolled in OME group, whereas children with adenoid hypertrophy (AH) who required adenoidectomy and individuals who required cochlear implantation (CI) were enrolled in AH and CI groups, respectively. Pepsinogen mRNA and protein levels were assessed by real-time fluorescence-based quantitative polymerase chain reaction and immunohistochemistry in adenoid specimens from the OME and AH groups. Pepsin and pepsinogen concentrations were evaluated by enzyme-linked immunosorbent assay in middle ear fluid and plasma from the OME and CI groups. RESULTS: The levels of pepsinogen protein expressed in cytoplasm of epithelial cells and clearance under epithelial cells in adenoid specimens from the OME group were significantly higher than those in the AH group. Furthermore, the concentrations of pepsin and pepsinogen in the OME group were 51.93±11.58 ng/mL and 728±342.6 ng/mL, respectively, which were significantly higher than those in the CI group (P<.001). In addition, the concentrations of pepsin in dry ears were significantly lower than those in serous and mucus ears in the OME group (F=22.77, P<.001).Finally, the concentration of pepsinogen in middle ear effusion was positively correlated with the expression intensity of pepsinogen protein in cytoplasm of epithelial cells (r=0.73, P<.05) in the OME group. CONCLUSIONS: Pepsin and pepsinogen in middle ear effusion are probably caused by LPR and may be involved in the pathogenesis of OME. LEVEL OF EVIDENCE: 3b.

[Chronic atrophic gastritis: morphological features and pepsin formation function].

The paper discusses a modern approach to chronic atrophic gastritis as a precancerous gastric condition. It deals with the role of Helicobacter pylori infection, development of disease, morphological changes taking place in gastric mucosa associated with chronic atrophic gastritis, and their importance for gastrocarcinogenesis. Recommendations are given on the strategies of management of precancerous gastric changes in mucosa as well as early prophylaxis of stomach cancer development from chronic atrophic gastritis associated with Helicobacter pylori infection.

Effect of fish oil on offensive and defensive factors in gastric ulceration in rats.

The effect of fish oil (FO) derived from Scomberoides commersonianus containing omega-3 polyunsaturated fatty acids was studied on gastric ulcers and as well as on offensive and defensive factors in gastric mucosal damage, following experimental gastric ulceration. FO significantly reduced the severity of ulceration in gastric ulcers induced by aspirin, cold-restraint stress (CRS), alcohol, and pylorus ligation. The results also indicated the potentiality of FO in maintaining the integrity of gastric mucosa by virtue of its effect on both offensive and defensive gastric mucosal factors. It decreased the offensive acid-pepsin secretion and augmented the defensive factors like mucin secretion, cellular mucus and life span of mucosal cells following pylorus ligation. FO significantly increased activity of anti-oxidant enzymes (catalase and glutathione peroxidase) and decreased lipid peroxidation in gastric mucosa of CRS rats. The study indicates the beneficial role of FO in gastric ulceration by inhibition of offensive mucosal factors and oxidative stress, and augmentation of defensive mucosal factors.

Molecular organization, expression and chromosomal localization of the mouse pronapsin gene.

Napsins have been identified only very recently as new aspartic proteinases of the pepsin family. Isolation, sequencing and functional analysis of the mouse genomic locus indicates that the organization of the pronapsin gene into nine exons is identical to that of other mammalian aspartic proteinase precursors, including pepsinogen. However, the additional C-terminal residues, which are a distinguishing feature of napsins, are encoded within exon 9 and not within an additional exon. Quantitation of pronapsin mRNA using RT-PCR indicates that the gene is transcribed in lung, kidney and spleen but not in heart. Regulation of gene expression was not influenced by the extent of CpG methylation but depended on the recognition of potential binding motifs in the promoter region by specific transcription factors such as YY-1. The single copy of the mouse pronapsin gene was located on chromosome 7. In humans, there are two pronapsin genes and, based on the mouse information, preliminary structures were deduced for these from sequences in the human genome databases. They appear to be located together on chromosome 19.

[Characteristics of enzymatic function of the stomach in gastroduodenal diseases in children with regard to gastric carcinogenesis]. / Osobennosti fermentativnoi funktsii zheludka pri gastroduodenal'noi patologii u detei v aspekte gastrokantserogeneza.

The paper deals with a description of the biochemical properties of the isoforms of pepsinogen pepsin of gastric mucosa and blood serum in children suffering from duodenal ulcerative disease as well as in atrophic and subtrophic lesions of gastric mucosa. Atrophic gastritis was found to involve an inhibited biosynthesis of the Ist fraction of pepsinogen while ulcerative-erosive lesions of the gastro-duodenal area--an increased level of the 3rd isoform of pepsinogen.

Influence of an intermittent compressive force on matrix protein expression by ROS 17/2.8 cells, with selective stimulation of osteopontin.

The purpose of this study was to determine the response of bone cells to physical stress. Intermittent compressive force (ICF) was applied to 13 kPa to subconfluent ROS 17/2.8 cells at 18 cycles/min. After 48 h of this application, the cells were labelled with [35S]-methionine or [32PO4]. Application of ICF over this time did not alter the synthesis of type I collagen, fibronectin or bone SPARC (osteonectin) compared to that of control cells. However, the activity of alkaline phosphatase was increased 1.5-fold, and the synthesis of a 32PO4-labelled, 75-kDa phosphoprotein, recognized as osteopontin by immunoprecipitation with specific antibodies, was increased 1.4-fold. Also, an increase in osteopontin mRNA starting within 12h of ICF application was observed. The selective increase in osteopontin expression associated with ICF may be important in the remodelling of bone tissues during growth and development and in response to functional forces.

Expression of human recombinant 72 kDa gelatinase and tissue inhibitor of metalloproteinase-2 (TIMP-2): characterization of complex and free enzyme.

The human 72 kDa gelatinase/type IV collagenase is a metalloproteinase that is thought to play a role in metastasis and angiogenesis. The 72 kDa progelatinase can be isolated from conditioned media as a complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2). To investigate 72 kDa gelatinase-TIMP-2 interactions and to compare the activity of the complex versus that of the free enzyme, we have expressed and purified human 72 kDa progelatinase and TIMP-2 as single proteins in a recombinant vaccinia virus mammalian cell expression system. The recombinant 72 kDa progelatinase was able to bind TIMP-2, and it digested gelatin and collagen type IV after activation by p-aminophenylmercuric acid (APMA). The specific activity of the recombinant free enzyme was 20-fold higher than the activity of an APMA-treated stoichiometric complex of recombinant 72 kDa progelatinase and TIMP-2. Also, TIMP-2 caused an 86% inhibition of activity when added to the activated enzyme at a 1:1 molar ratio. Activation of the free recombinant 72 kDa progelatinase yielded the 62 kDa species and two fragments of 46 and 35 kDa that cross-reacted with monoclonal antibodies to the 72 kDa proenzyme. TIMP-2 inhibited the conversion of the recombinant proenzyme to the 62 kDa species and the appearance of the 45 and 35 kDa bands. These results suggest that TIMP-2 is not only a potent inhibitor of the activated enzyme but also prevents the generation of low-molecular-mass species and full enzymic activity from the zymogen.

Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines.

Collagenase production by rodent osteoblasts in response to calciotropic hormones has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby exposing the underlying mineralized matrix to osteoclastic action. Many studies suggest, however, that this model might not apply to bone resorption in the human. Human osteoblasts have been shown to produce gelatinase-A (72 kDa) and TIMP-1 (tissue inhibitor of metalloproteinases), but previous investigators have been unable to demonstrate the synthesis of collagenase by human osteoblasts either constitutively or in response to bone resorptive agents. In the present study the ability of human osteoblasts to produce the matrix metalloproteinases (MMPs) collagenase, gelatinase and stromelysin, and their specific inhibitors TIMPs-1 and 2, was examined using highly sensitive and specific antisera and by zymography. Semi-quantitative histomorphometric data showed that cells cultured on either glass or a type I collagen substratum constitutively synthesized gelatinase-A and TIMP-1. On type I collagen, however, a small proportion of unstimulated cells produce both collagenase (7%) and gelatinase-B (95 kDa; 3%). Treatment of cells with either parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), or partially purified mononuclear cell conditioned medium (MCM), stimulated the synthesis of collagenase, gelatinase-B and stromelysin; MCM was 2- to 3-fold more potent than either PTH or 1,25(OH)2D3. Zymography using SDS/PAGE on conditioned media from cells cultured on type I collagen films revealed the presence of active gelatinase-A and that MCM stimulated progelatinase-B synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of neutral proteinase and prostanoid production in bovine nasal chondrocytes by interleukin-1 and tumor necrosis factor alpha: modulation of these cellular responses by interleukin-6 and platelet-derived growth factor.

We have previously reported that recombinant human interleukin-1 (IL-1) stimulates matrix erosion in bovine nasal cartilage explants (R. J. Smith et al., Inflammation 13, 367-382, 1989). This action of IL-1 is believed to be caused by matrix-degrading neutral proteinases produced by activated chrondrocytes. Accordingly, we investigated the effects of recombinant human interleukin-1 alpha (IL-1 alpha), recombinant human interleukin-1 beta (IL-1 beta), and recombinant human tumor necrosis factor alpha (TNF alpha) on bovine nasal chondrocyte (BNC) responsiveness. IL-1 alpha and IL-1 beta stimulated a time (0-72 hr) and concentration-dependent (0.01-10 ng/ml) production of collagenase, gelatinase, caseinase, and prostaglandin E2 (PGE2) in BNC monolayer cultures. Neutral proteinase and PGE2 production by BNC was also induced by TNF alpha (0.2-200 ng/ml) in a time-dependent (0-72 hr) manner. Recombinant human interleukin-6 (IL-6) caused a concentration-dependent (6-200 ng/ml) potentiation of IL-1-stimulated neutral proteinase and PGE2 production by BNC. However, recombinant human platelet-derived growth factor homodimer BB suppressed BNC responsiveness to IL-1. A recombinant human IL-1 receptor antagonist protein inhibited BNC activation by IL-1 but not TNF alpha.

Induction of 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines.

Gelatinases/type IV collagenases have been shown to be involved in tumor invasion and metastasis. In this study, we examined the effect of culture medium pH on the secretion of the gelatinases from mouse B16 melanoma cell lines and human tumor cell lines using zymography analysis. The highly metastatic clone F10 of B16 melanoma did not secrete any gelatinase in neutral culture media (pH 7.1-7.3), whereas it secreted a high level of a 103-kDa gelatinase in an initial pH range of 5.4-6.1. The addition of an excess amount of glucose into a neutral culture medium also induced the gelatinase secretion from the cells by decreasing the medium pH during incubation. The extent of the acid-induced gelatinase secretion by the B16 melanoma cell lines was in the order of BL6 greater than F10 greater than F1 much greater than the parent B16 line, in good agreement with the order of their metastatic potentials. Two human cell lines (A549 and HT1080) secreted a higher level of a 90-kDa gelatinase at pH 6.8 compared with pH 7.3. The acid-induced gelatinase secretion from B16-F10 cells was blocked by cycloheximide, indicating that the enzyme induction was due to de novo synthesis. When in vitro tumor cell invasion was assayed in Boyden chambers, B16-F10 cells incubated in an acidic medium exerted a more active migration through type IV collagen gel than those in a neutral medium. These results suggest that the acidic environment formed around tumor tissues may be an important factor in invasion and metastasis of some types of tumors.

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.

Constitutive expression of a 92-kD gelatinase (type V collagenase) by rheumatoid synovial fibroblasts and its induction in normal human fibroblasts by inflammatory cytokines.

Synovial fibroblasts freshly isolated from the rheumatoid joint are characterized by their marked connective tissue degradative ability. This phenotype includes the ability to secrete large amounts of the matrix-degrading metalloproteinases, collagenase, and stromelysin. We have found that another aspect of this phenotype is the constitutive expression at both protein and mRNA levels of a 92-kD gelatinolytic metalloproteinase, which is not secreted by normal dermal or lung fibroblasts and is immunologically cross-reactive with a type V collagenase expressed by activated macrophages and neutrophils. Expression of this 92-kD metalloproteinase confers upon the fibroblasts the capacity to degrade collagenase- and stromelysin-resistant interstitial elements, such as collagen types IV, V and XI. In contrast to the 92-kD metalloproteinase, a 68-kD gelatinase (type IV collagenase) was expressed by all fibroblast types studied, indicating that its regulation is distinct from that of the 92-kD gelatinase. To identify what cytokines may be important in the induction of the rheumatoid synovial phenotype, including expression of the 92-kD gelatinase, we exposed normal dermal fibroblasts to a number of cytokines including many known or considered likely to be present in rheumatoid synovial fluid and tissue. Although IL-1 beta, tumor necrosis factor-alpha, lymphotoxin, platelet-derived growth factor, and basic fibroblast growth factor were capable of stimulating fibroblasts to secrete collagenase, only tumor necrosis factor-alpha, lymphotoxin, and IL-1 beta were able to induce expression of the 92-kD gelatinase, demonstrating discordant regulation of the two metalloproteinases. Expression of the 68-kD gelatinase was independent of that of the 92-kD gelatinase, as demonstrated at the protein and mRNA levels. Late passage rheumatoid synovial fibroblasts, which no longer constitutively expressed the 92-kD gelatinase, displayed an accentuated response to IL-1 beta when compared to normal dermal fibroblasts. Thus, in addition to IL-1 beta, tumor necrosis factor-alpha or lymphotoxin may contribute to the expression of a specific rheumatoid synovial phenotype in vivo that is associated with progressive matrix destruction.

Human melanoma cell invasion is inhibited in vitro by swainsonine and deoxymannojirimycin with a concomitant decrease in collagenase IV expression.

A 48 h pretreatment of two malignant and invasive human melanoma cell lines with either swainsonine (an inhibitor of Golgi alpha-mannosidase II) or deoxymannojirimycin (a Golgi alpha-mannosidase I inhibitor) resulted in a dose-dependent decrease in the cells' ability to invade a reconstituted basement membrane in vitro. This effect was reversible within 48 h of removing the drugs. Treatment with either drug resulted in both cell lines being more resistant to the cytotoxic effects of the lectin leukoagglutinin (PHA-L) and more sensitive to the lectin concanavalin A which indirectly indicated a change in the cell surface oligosaccharide composition and structure consistent with the known effects of these drugs on N-linked oligosaccharide processing. A 25-33% decrease was noted in the adhesion of treated cells to either a reconstituted basement membrane or human umbilical vein endothelial cell monolayer while no change was measured in the cells' proliferative rates. A correlative decrease was observed, however, in the expression of human type IV collagenase mRNA which was recovered within 48 h of removing the drugs. These results suggest that a correlation exists between the drug-induced changes in the cell surface oligosaccharide composition and structure with a concomitant decrease in the mRNA and secreted levels of type IV collagenase and the ability of these cells to invade.

Lipase and pepsin activities in the stomach mucosa of the suckling dog.

The origin of preduodenal lipases was investigated in the suckling dog. In this species, gastric lipase is the main (or only) preduodenal lipase (activity range 1.03-8.32 U/mg protein), lingual-lipase activity being absent or amounting to traces only (0.77-2.3 mU/mg tissue). The localization of lipase activity in the stomach was mapped and compared to that of pepsin. The data show that the highest lipase activity was found in biopsy specimens of gastric mucosa from the cardia area (5.32 +/- 1.29-8.32 +/- 0.93 U/mg protein), and the lowest in the antrum (1.03 +/- 0.16-1.94 +/- 0.43 U/mg protein). Activity was also significantly higher in the mucosa along the greater rather than the lesser curvature of the stomach. Pepsin activity was highest in the cardia and gastric body areas (26.2 +/- 0.89-89 +/- 23.61 and 26.83 +/- 15.98-69.51 +/- 9.82, respectively). Contrary to lipase activity, considerable pepsin activity was present in the antrum (18.17 +/- 4.12-23.07 +/- 5.60 U/mg protein). No difference in pepsin activities was found in the greater as compared to the lesser curvature. The data show similar origin and tissue distribution of gastric digestive enzymes in the suckling dog and human infant. The role of the newborn dog as an animal model for fat digestion in the human infant is discussed.

Concanavalin A produces a matrix-degradative phenotype in human fibroblasts. Induction and endogenous activation of collagenase, 72-kDa gelatinase, and Pump-1 is accompanied by the suppression of the tissue inhibitor of matrix metalloproteinases.

The lectin concanavalin A (ConA) causes fibroblasts to acquire an arborized morphology and to express elevated levels of collagenase. The temporal and mechanistic aspects of ConA regulation of matrix metalloproteinases (MMPs) and the tissue inhibitor of matrix metalloproteinases (TIMP) were characterized in early passage human fibroblasts. Collagenase (MMP-1), measured by functional assays in the absence of TIMP and also as immunoprecipitated [35S]methionine-labeled protein, was increased 10-20-fold following ConA (20 micrograms/ml, 2 x 10(-7) M) treatment for 24-72 h, with active collagenase comprising approximately 20% of the total collagenase activity. By comparison, MMP-2 (72-kDa gelatinase; molecular mass, 72 kDa, +dithiothreitol; 66 kDa, -dithiothreitol), analyzed by enzymography and following affinity purification, was increased less than 2-fold by ConA and was present entirely as an activated, 61-kDa (+dithiothreitol; 59 kDa, -dithiothreitol) form. Northern hybridization analyses revealed that ConA elevated the steady-state mRNA levels for MMPs; collagenase mRNA increased approximately 16-fold, MMP-2 increased 2-fold, and Pump-1, a recently described MMP gene, was induced. Concomitantly, a 10-fold reduction in TIMP protein and mRNA levels by ConA occurred. In comparison, 12-O-tetradecanoylphorbol-13-acetate (50 ng/ml, 8 x 10(-8) M), which also stimulates collagenase expression strongly (greater than 30-fold), elevated TIMP protein and mRNA levels (2- and 3-fold, respectively) and did not affect MMP-2 expression. The changes in MMP and TIMP mRNA levels induced by ConA were blocked by the protein synthesis inhibitor cycloheximide, and the half-lives of collagenase and MMP-2 mRNAs (53 and 46 h, respectively) were unaffected, indicating that ConA exerts its effects transcriptionally, through pathways requiring de novo protein synthesis. Increased transcription of the mmp genes was confirmed by nuclear run-on analyses; mmp-1 transcription was increased by greater than 25-fold, mmp-2 by approximately 3-fold, and Pump-1 by approximately 7-fold. In contrast, Timp gene transcription was reduced by approximately 80%, revealing reciprocal regulation of MMPs and TIMP during the induction of a resorptive cell phenotype. Decreased amounts of collagen and fibronectin, but not of SPARC (secreted protein, acidic and rich in cysteine) in the conditioned medium was the result of MMP activity since steady-state mRNA levels and transcription of the respective matrix protein genes were unaffected by ConA.

Induction and stimulation of 92-kDa gelatinase/type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor alpha.

Production of a 92-kDa gelatinase/type IV collagenase and tissue inhibitor of metalloproteinases (TIMP) was investigated with human sarcoma cell lines. Among the cytokines and growth factors examined, only human recombinant tumor necrosis factor alpha (TNF alpha) induced and stimulated the proteinase with concomitant increase in TIMP expression, but matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) expression was unchanged. These data suggest that gene expression of the two metalloproteinases is regulated in a different fashion and TNF alpha may be important to allow cancer cells to be more invasive and metastatic.