Abstract: FR-PO620

An N-Terminal Truncated Intracellular Isoform of Matrix Metalloproteinase-2 Is Induced by Hyperglycemia and Activates a Primary Innate Immune Response In Vitro

Session Information

Category: Diabetes

  • 501 Diabetes Mellitus and Obesity: Basic - Experimental

Authors

  • Song, Sang Heon, Pusan National University Hospital, Busan, Korea (the Republic of)
  • Park, In seong, Pusan National University Hospital, Busan, Korea (the Republic of)
  • Han, Miyeun, Pusan National University Hospital, Busan, Korea (the Republic of)
  • Rhee, Harin, Pusan National University Hospital, Busan, Korea (the Republic of)
  • Seong, Eun Young, Pusan National University Hospital, Busan, Korea (the Republic of)
  • Lee, Dong Won, Pusan National University School of Medicine, Yangsan, Korea (the Republic of)
  • Lee, Soo Bong, Pusan National University School of Medicine, Yangsan, Korea (the Republic of)
  • Kwak, Ihm Soo, Pusan National University Hospital, Busan, Korea (the Republic of)
  • Lovett, David H., University of California San Francisco, San Francisco, California, United States
Background

We have reported that matrix metalloproteinase-2 (MMP-2) exists in two discrete isoforms. The first consists of the classical full length isoform (FL-MMP-2) which is secreted as a latent proenzyme. A second novel isoform is generated by oxidative stress-mediated activation of an alternate promoter in the distal first intron of the MMP-2 gene. This results in synthesis of an N-terminal truncated isoform (NTT-MMP-2) that is intracellular, enzymatically active and concentrated within mitochondria. NTT-MMP-2 triggers mitochondrial-nuclear stress signaling via NF-kB transcriptional cascades. Renal proximal tubule-specific transgenic expression of the NTT-MMP-2 isoform results in regulated necrosis, activation of innate immunity and enhanced sensitivity to ischemia/reperfusion injury. In this study we examined the relationship between hyperglycemia and the induction of NTT-MMP-2 and innate immunity genes using the human HK2 proximal tubule cell line.

Methods

We manufactured the specific siRNA for NTT-MMP-2 and tested the inhibitory efficacy of NTT-MMP-2 for innate immunity-related genes using quantitative PCR in HK2 cells with high glucose stimulation.

Results

High glucose medium (30 mM) induced a 1.65±0.06 and a 3.20±0.18 fold increase in FL-MMP-2 and NTT-MMP-2 synthesis as determined by qPCR, respectively (p<0.05 for each). High glucose medium also resulted in statistically significant increases in the expression of the innate immunity genes IFIT1, IRF7, IL6 and CXCL1 including NF-kB. To determine the relationship between high glucose medium enhanced NTT-MMP-2 and innate immune gene expression we developed an NTT-MMP-2 specific siRNA that targets the unique 5’UTR of this gene. Selective siRNA targeting inhibited high glucose mediated NTT-MMP-2 expression to 0.81±0.75 fold (p<0.01), with no effect on FL-MMP-2 expression (1.33±0.28 fold, p>0.05) compared with negative control. The selective NTT-MMP-2 siRNA suppressed high glucose mediated NF-kB expression, as well as IFIT1 ,IRF7, IL6 and CXCL1.

Conclusion

We conclude that selectively targeting NTT-MMP-2 with a siRNA approach offers a therapeutic approach for reduction of tubular epithelial cell necrosis and activation of innate immunity in the setting of diabetes mellitus.

Funding

  • Government Support - Non-U.S.