Role of TACE in LPS‐induced Production of Soluble Tumor Necrosis Factor‐α in Astrocytes

Abstract

The pro‐inflammatory cytokine tumor necrosis factor alpha (TNF‐α) in the brain has been implicated in the neurohumoral activation in cardiovascular diseases like heart failure and hypertension. TNF‐α is originally synthesized as a membrane bound protein, the transmembrane TNF‐α (tmTNF‐α). TNF‐α converting enzyme (TACE) proteolytically cleaves the extracellular domain of tmTNF‐α) to release the fully functional form of soluble TNF‐α (sTNF‐α), the major form that contributes to the sympathetic excitation‐induced by cytokine TNF‐α. Our previous work has demonstrated a significant contribution of cytokine synthesis within the central nervous system in mediating neurohumoral excitation and the progression of heart failure in rats, and that both TNF‐α and TACE are increased within cardiovascular regulatory brain nuclei in rats with heart failure. However, the molecular mechanisms regulating TNF‐α and TACE expression within the brain and the specific cell types contributing to this process are currently unknown. The present study sought to determine whether astrocytes might contribute to central nervous system inflammation as a significant source of TNF‐α and TACE. We isolated and cultured astrocytes from male and female neonatal rat brains. Cells were maintained in culture for 28 days prior to treatment with lipopolysaccharide (LPS) alone or in combination with a matrix metalloproteinase inhibitor. Cells and conditioned media were collected after 8 hours to measure RNA expression as well as TNF‐α secretion. Primary astrocytes expressed TACE mRNA at baseline, and LPS increased mRNA levels of TNF‐α (2.8±0.6 vs 3.8±2.1‐fold change), interleukin‐1β (IL‐1β) (19.9±12.3 vs 22.2±7.6‐fold change), and TACE (1.8±0.2 vs 2.2±0.3‐fold change) to a similar extent in astrocytes from male and female rats. Neither the specific TACE inhibitor TAPI‐1 nor the broad range metalloproteinase inhibitor GM6001 significantly attenuated the LPS‐induced increase in TNF‐α at the mRNA level, and in fact we observed a trend towards increased TNF‐α expression specifically in astrocytes from male animals co‐treated with LPS and either inhibitor. This was in contrast to protein expression of sTNF‐α. LPS significantly increased TNF‐α secretion into the media in astrocytes from male (677±28 vs 5±1pg/ml) and female (658±42 vs 6±4pg/ml) animals to a similar extent. Interestingly, this was attenuated by treatment with TAPI‐1 (169±14 male and 108±11pg/ml female) but not GM6001 (526±51 male and 667±44pg/ml female). However, the LPS‐induced increase in TACE expression was attenuated in cells treated with GM6001 but not those treated with TAPI‐1. These data suggest that astrocytes in the brain may contribute to the production of cytokines and TACE in pathophysiological conditions, and TACE plays an important role in releasing the inflammatory sTNF‐α from its precursor tmTNF‐α.Support or Funding InformationSupported by R01‐HL139521 and T32 HL007121.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.