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Murine fibroblasts synthesize and secrete kininogen in response to cyclic-AMP, prostaglandin E2 and tumor necrosis
M Takano1, K Yokoyama, K Yayama
1Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Japan.
Abstract:
Fibroblasts prepared from the meninges of newborn mice or from mouse embryos, as well as fibroblast L929 cells, secreted an immunoreactive material (ir-kininogen) against rabbit anti-mouse low-molecular-weight kininogen antibody in response to dibutyryl cAMP. Western blots using a bradykinin-directed monoclonal, as well as a polyclonal anti-mouse low-molecular-weight kininogen antibody, showed that ir-kininogen had a molecular weight of 80,000 and that it contained a kinin moiety. N-terminal amino acid sequence of the ir-kininogen was consistent with that of mouse L-kininogen. The ir-kininogen produced by fibroblasts released a kinin by incubating with trypsin and mouse submandibular gland kallikrein, and it was identified as bradykinin by means of high-performance liquid chromatography, indicating that mouse fibroblasts produce and secrete a kininogen. Forskolin, prostaglandin E2 and tumor necrosis factor alpha stimulated the production of ir-kininogen by meningeal fibroblasts, whereas neither dibutyryl cAMP nor these agents influenced kininogen production by mouse hepatocytes in primary cultures. These results demonstrated that fibroblasts are a source of kininogen in the mouse, and that it is regulated by the inflammatory mediators, prostaglandin E2 and tumor necrosis factor. Therefore locally produced kininogen is implicated in pathogenesis of inflammation.
Insights
Mouse fibroblasts produce and secrete kininogen, a protein involved in inflammation. Production is stimulated by inflammatory mediators like prostaglandin E2 and tumor necrosis factor alpha.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Kininogen is a precursor to kinins, potent vasoactive peptides.
- The cellular sources and regulation of kininogen production, particularly in inflammatory contexts, require further elucidation.
Purpose of the Study:
- To investigate whether mouse fibroblasts produce and secrete kininogen.
- To identify the molecular characteristics of fibroblast-derived kininogen.
- To explore the regulation of fibroblast kininogen production by inflammatory mediators.
Main Methods:
- Fibroblast cultures (meningeal, embryonic, L929 cell line) were stimulated with dibutyryl cAMP.
- Western blotting was performed using antibodies against mouse low-molecular-weight kininogen and bradykinin.
- N-terminal amino acid sequencing and high-performance liquid chromatography (HPLC) were used for identification.
- Primary mouse hepatocyte cultures were used for comparative studies.
Main Results:
- Fibroblasts secreted an 80,000 MW immunoreactive kininogen (ir-kininogen) in response to dibutyryl cAMP.
- The ir-kininogen contained a kinin moiety and its N-terminal sequence matched mouse L-kininogen.
- Fibroblast-derived ir-kininogen released bradykinin upon enzymatic treatment.
- Prostaglandin E2 and tumor necrosis factor alpha stimulated ir-kininogen production in meningeal fibroblasts, but not in hepatocytes.
Conclusions:
- Mouse fibroblasts are a significant source of kininogen.
- Fibroblast kininogen production is regulated by inflammatory mediators, including prostaglandin E2 and tumor necrosis factor alpha.
- Locally produced fibroblast kininogen is potentially involved in the pathogenesis of inflammation.