Research Paper Volume 13, Issue 3 pp 4452—4467

Modeling paraquat-induced lung fibrosis in C. elegans reveals KRIT1 as a key regulator of collagen gene transcription

Conserved roles of Krit1 and Nrf2 in collagen transcription in human lung fibroblasts. (A) A Volcano Plot showing collagen genes expression were down-regulated in krit1-knockdown mouse primary brain microvascular endothelial cells. Dataset GSE85657 was downloaded from NCBI and analyzed by Rstudio. Red data points indicate collagen transcripts and blue other transcripts detected by RNA sequencing. Arrows point to top-ranked transcripts selected for further study. (B, C) Paraquat up-regulated COL27A1 and COL28A1 expression in human lung fibroblasts. MRC-5 cells were transfected with KRIT1 and NRF2 siRNAs and treated with 300 μM of paraquat for 48 hours. Total mRNA was purified and reverse transcribed for RT-qPCR analysis. Error bars indicate the standard deviation of 3 experiments. P values were obtained by two tailed, paired student’s t-test (*PD) KRIT1 and NRF2 knockdown inhibit paraquat induction of collagen transcription. Data from (B, C) were transformed to fold changes by paraquat treatment and statistically tested by two tailed, paired student’s t-test (*PF) KEAP1 knockdown up-regulated collagen transcription in human lung fibroblasts. Experiments were conducted as in (B, C) except that KEAP1 siRNA was used. Error bars indicate the standard deviation of 3 experiments. P values were obtained by two tailed, paired student’s t-test (*PG) KEAP1 knockdown up-regulated collagen transcription preferentially in KRIT1-knockdown cells. Data from (D, E) were transformed into fold change by KEAP1 knockdown and statistically tested by two tailed, paired student’s t-test (*PH) A working model showing KRIT1/KRI-1 regulation of collagen transcription. Paraquat treatment could generate protective signals through KRIT1/KRI-1, which in turn activates Nrf2/SKN-1 pathway to promote collagen transcription. Up-regulation of collagen may serve to protect cells and organisms from paraquat toxicity.

Figure 5. Conserved roles of Krit1 and Nrf2 in collagen transcription in human lung fibroblasts. (A) A Volcano Plot showing collagen genes expression were down-regulated in krit1-knockdown mouse primary brain microvascular endothelial cells. Dataset GSE85657 was downloaded from NCBI and analyzed by Rstudio. Red data points indicate collagen transcripts and blue other transcripts detected by RNA sequencing. Arrows point to top-ranked transcripts selected for further study. (B, C) Paraquat up-regulated COL27A1 and COL28A1 expression in human lung fibroblasts. MRC-5 cells were transfected with KRIT1 and NRF2 siRNAs and treated with 300 μM of paraquat for 48 hours. Total mRNA was purified and reverse transcribed for RT-qPCR analysis. Error bars indicate the standard deviation of 3 experiments. P values were obtained by two tailed, paired student’s t-test (*P<0.05, **P<0.01, ***P<0.001). (D) KRIT1 and NRF2 knockdown inhibit paraquat induction of collagen transcription. Data from (B, C) were transformed to fold changes by paraquat treatment and statistically tested by two tailed, paired student’s t-test (*P<0.05, **P<0.01). (F) KEAP1 knockdown up-regulated collagen transcription in human lung fibroblasts. Experiments were conducted as in (B, C) except that KEAP1 siRNA was used. Error bars indicate the standard deviation of 3 experiments. P values were obtained by two tailed, paired student’s t-test (*P<0.05, ***P<0.001). (G) KEAP1 knockdown up-regulated collagen transcription preferentially in KRIT1-knockdown cells. Data from (D, E) were transformed into fold change by KEAP1 knockdown and statistically tested by two tailed, paired student’s t-test (*P<0.05). (H) A working model showing KRIT1/KRI-1 regulation of collagen transcription. Paraquat treatment could generate protective signals through KRIT1/KRI-1, which in turn activates Nrf2/SKN-1 pathway to promote collagen transcription. Up-regulation of collagen may serve to protect cells and organisms from paraquat toxicity.