Both asparagine and proline are required to decrease <i>Sestrin2</i> mRNA levels via ATF4 reduction and regulate collagen type I alpha 1 chain production and the proliferation of quiescent RI‑T hepatic stellate cells

Sestrin 2 (SESN2) is a conserved protein whose expression is upregulated under various cellular stresses including hepatic injury. In the injured liver, hepatic stellate cells (HSCs) become activated and produce collagen, contributing to fibrosis; however, SESN2 overexpression has been shown to suppress collagen synthesis. Amino acids are known to influence SESN2 expression; however, their specific effects remain unclear. In the present study, it was investigated whether specific amino acids regulate SESN2 expression and the function of quiescent RI-T HSCs, which are responsible for collagen production, using reverse transcription-quantitative PCR, western blotting, and cell proliferation assay. It was found that supplementation with asparagine (Asn) and proline (Pro) (AP), both non-essential amino acids, led to a complete reduction in <i>SESN2</i> and <i>activating transcription factor 4 (ATF4)</i> mRNA levels after 5 h of incubation. Additionally, AP partially reduced <i>collagen type I α1 (COL1A1)</i> mRNA levels. However, knockdown of SESN2 or ATF4 resulted in a more substantial reduction in <i>COL1A1</i> mRNA levels than the supplementation with AP. These results suggest that SESN2, which is induced by amino acid insufficiency, contributes to the upregulation of <i>COL1A1</i> mRNA levels and that AP may increase <i>COL1A1</i> mRNA levels through pathways independent of SESN2. The COL1A1-inducing effect of SESN2 contrasted with the inhibitory effect of SESN2 on activated HSCs. In long-term cultures, AP supplementation increased <i>COL1A1</i> mRNA and protein levels, as well as RI-T cell proliferation, while <i>SESN2</i> and <i>ATF4</i> mRNA levels remained suppressed. These findings suggested that the absence of AP induces relative amino acid starvation, leading to increased ATF4/SESN2 expression. By contrast, long-term AP supplementation alleviated this stress, promoting cell proliferation and COL1A1 synthesis. The present results indicate that SESN2 function in quiescent HSCs may differ from its role in activated cells, providing new insights into its regulatory mechanisms in collagen production.