Mitophagy is involved in sepsis-induced acute lung injury (ALI). Bcl-2 family proteins play an important role in mitochondrial homeostasis. However, whether targeting Bcl-2 proteins (Bcl-2 and Bad) could influence mitophagy in ALI remains unclear. In this study, lipopolysaccharide (LPS) was used to induce injury in A549 cells and ALI in mice. LPS treatment resulted in elevated cell apoptosis, enhanced mitophagy, decreased Bcl-2 expression, increased Bad expression, and activation of PINK1/Parkin signaling in cells and lung tissues. Both Bcl-2 overexpression and Bad knockdown attenuated LPS-induced injury, inhibited cell apoptosis and mitophagy, and improved survival. Atg5 knockout (KO) inhibited LPS-induced cell apoptosis. Furthermore, Bcl-2 proteins regulated mitophagy by modulating the recruitment of Parkin from the cytoplasm to mitochondria via direct protein-protein interactions. These results were further confirmed in Park2 KO cells and Park2^-/- mice. This is the first study to demonstrate that Bcl-2 proteins regulated mitophagy in LPS-induced ALI via modulating the PINK1/Parkin signaling pathway, promoting new insights into the mechanisms and investigation of therapeutic strategies for a septic patient with ALI.

Vascular calcification is a highly regulated biological process similar to bone formation involving osteogenic differentiation of vascular smooth muscle cells (VSMCs). Hyaluronan (HA), a major structural component of the extracellular matrix in cartilage, has been shown to inhibit osteoblast differentiation. However, whether HA affects osteogenic differentiation and calcification of VSMCs remains unclear. In the present study, we used in vitro and ex vivo models of vascular calcification to investigate the role of HA in vascular calcification. Both high and low molecular weight HA treatment significantly reduced calcification of rat VSMCs in a dose-dependent manner, as detected by alizarin red staining and calcium content assay. Ex vivo study further confirmed the inhibitory effect of HA on vascular calcification. Similarly, HA treatment decreased ALP activity and expression of bone-related molecules including Runx2, BMP2 and Msx2. By contrast, inhibition of HA synthesis by 4-methylumbelliferone (4MU) promoted calcification of rat VSMCs. In addition, adenovirus-mediated overexpression of HA synthase 2 (HAS2), a major HA synthase in VSMCs, also inhibited calcification of VSMCs, whereas CRISPR/Cas9-mediated HAS2 knockout promoted calcification of rat A10 cells. Furthermore, we found that BMP2 signaling was inhibited in VSMCs after HA treatment. Recombinant BMP2 enhanced high calcium and phosphate-induced VSMC calcification, which can be blocked by HA treatment. Taken together, these findings suggest that HA inhibits vascular calcification involving BMP2 signaling.

BACKGROUND: Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD), and the underlying mechanisms remain elusive. Several studies have indicated an important role of protein ubiquitination in vascular calcification. However, the role of E3 ubiquitin ligases in vascular calcification remains poorly understood. METHODS: Calcification of vascular smooth muscle cells (VSMCs) was induced by high phosphate. CKD mouse model was induced by an adenine diet, and CKD rat model was established using 5/6 nephrectomy method. Adenovirus, siRNA, adeno-associated virus, and smooth muscle cell–specific TRIM16 (tripartite motif 16) knockout mice were used to investigate the role of TRIM16 in vascular calcification. Immunoprecipitation–mass spectrometry, and ubiquitination assay were used to dissect how TRIM16 regulates vascular calcification. RESULTS: Bioinformatic analysis suggested that E3 ubiquitin TRIM16 could be a key modulator of vascular calcification. TRIM16 expression was increased during vascular calcification. Overexpression of TRIM16 exacerbated VSMC calcification and aortic calcification of CKD rats. By contrast, the knockdown of TRIM16 alleviated VSMC calcification and aortic calcification in CKD rats. Moreover, the deletion of TRIM16 inhibited VSMC calcification and aortic calcification in VitD3-overloaded mice and CKD mice. Mechanistically, immunoprecipitation–mass spectrometry revealed that DAB2 (disabled homolog 2) was the potential downstream target of TRIM16. Coimmunoprecipitation showed that the SPRY domain of TRIM16 interacted with the proline-rich domain of DAB2. Moreover, ubiquitination assay revealed that the SPRY domain of TRIM16 is required for TRIM16-mediated K63-linked ubiquitination of DAB2 at K656 residue. Of note, the knockdown of DAB2 inhibited TRIM16-induced VSMC calcification. Furthermore, knockdown of DAB2 antagonized endocytosis of integrin β1 and subsequent activation of FAK (focal adhesion kinase)-STAT3 (signal transduction and transcriptional activation factor 3) signaling induced by overexpression of TRIM16. CONCLUSIONS: Our study for the first time demonstrates that E3 ubiquitin ligase TRIM16 catalyzes K63-ubiquitination of the endocytic adaptor DAB2, leading to endocytosis of integrin β1 and activation of FAK-STAT3 signaling, and subsequently exacerbates vascular calcification, suggesting that TRIM16-DAB2 axis is a promising therapeutic target for vascular calcification.

Heat shock protein B8 (HSPB8) has been recently discovered to be participated in the regulation of tumor progression. However, the function of HSPB8 in intrahepatic cholangiocarcinoma (ICC) has not yet been elucidated. This study studied the function of HSPB8 in ICC progression. ICC patients (n = 150) were enrolled. The relationship between clinicopathological characteristics and HSPB8 expression was analyzed. RBE cells were transfected and treated by 3-MA. The RBE cells morphology was observed under a transmission electron microscope. Cell counting kit-8 assay, wound healing assay and Transwell experiment was conducted to detect RBE cells proliferation, migration and invasion. Quantitative reverse transcription-polymerase chain reaction, immunohistochemistry, Western blot and immunofluorescence were used for genes detection in clinical tissues and RBE cells. HSPB8 was up-regulated in ICC tissues than that in adjacent normal tissues. High HSPB8 expression in ICC indicated poor prognosis of patients. HSPB8 expression was mainly expressed in cell cytoplasm and aberrantly increased in RBE cells (P < 0.01). HSPB8 up-regulation promoted RBE cells proliferation, migration and invasion (P < 0.05). HSPB8 down-regulation reduced RBE cells proliferation, migration and invasion (P < 0.01). HSPB8 overexpression facilitated Vimentin expression, LC3-II/LC3-I ratio and inhibited E-cadherin, p62 expression in RBE cells (P < 0.05). Treatment of 3-MA partially reversed HSPB8 promotion on RBE cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) (P < 0.05 or P < 0.01). HSPB8 promoted ICC progression by enhancing EMT and autophagy. HSPB8 might be an effective target for ICC treatment.