CD44 is involved in liver regeneration through enhanced uptake of extracellular cystine

Dear Editor, Liver regeneration triggered by fulminant liver damage accompanies the proliferation of hepatic progenitor cells (HPCs). Thus, there has been considerable interest in identifying specific HPC markers. However, the physiological role of HPC markers during liver regeneration is not fully understood. Herein, we identify the mechanistic roles of CD44, a cell surface marker for hepatic regeneration,1 in HPC proliferation and redox homeostasis. The Gene Expression Omnibus data revealed an increase in CD44 expression after partial hepatectomy (Figure 1A). Remnants of the mouse left hepatic lobe, which had undergone resection of more than 80% of its volume, showed significant elevation of CD44 expression. CD44 expression peaked at 10 days after the resection, at which time, the level of the proliferation marker, proliferating cell nuclear antigen (PCNA), was the highest (Figure 1B, Figure S1A). We isolated primary hepatocytes from the liver 10 days after resection and confirmed the increase of CD44 expression in the hepatocytes (Figure 1C–E). Acetaminophen (APAP)-induced acute liver injury also increased hepatic CD44 expression.2 The expression of CD44 peaked at 48 h after APAP treatment (Figure 1F). Primary hepatocytes from mice after APAP injection showed higher levels of PCNA and CD44 than did hepatocytes from phosphate-buffered saline (PBS)-injected mice (Figure 1G,H). Approximately 8% of the hepatocytes from APAPinjectedmice constituted a discrete population with a high CD44 level (Figure 1I). The subpopulation of hepatocytes also highly expressed PCNA and anHPCmarker,3 EpCAM (Figure 1J,K, Figure S1B). To further identify the CD44expressing hepatocytes as HPCs, we verified their proliferation capacity. Considering the role of CD44 as an adhesion molecule that binds to hyaluronic acid (HA), we isolated small hepatocytes (Figure S1C) and cultured them on an HA-coated dish.4 The small hepatocytes isolated fromAPAP-injectedmice proliferated and formed colonies

Dear Editor, Liver regeneration triggered by fulminant liver damage accompanies the proliferation of hepatic progenitor cells (HPCs). Thus, there has been considerable interest in identifying specific HPC markers. However, the physiological role of HPC markers during liver regeneration is not fully understood. Herein, we identify the mechanistic roles of CD44, a cell surface marker for hepatic regeneration, 1 in HPC proliferation and redox homeostasis.
The Gene Expression Omnibus data revealed an increase in CD44 expression after partial hepatectomy ( Figure 1A). Remnants of the mouse left hepatic lobe, which had undergone resection of more than 80% of its volume, showed significant elevation of CD44 expression. CD44 expression peaked at 10 days after the resection, at which time, the level of the proliferation marker, proliferating cell nuclear antigen (PCNA), was the highest ( Figure 1B, Figure S1A). We isolated primary hepatocytes from the liver 10 days after resection and confirmed the increase of CD44 expression in the hepatocytes ( Figure 1C-E). Acetaminophen (APAP)-induced acute liver injury also increased hepatic CD44 expression. 2 The expression of CD44 peaked at 48 h after APAP treatment ( Figure 1F). Primary hepatocytes from mice after APAP injection showed higher levels of PCNA and CD44 than did hepatocytes from phosphate-buffered saline (PBS)-injected mice ( Figure 1G,H).
Approximately 8% of the hepatocytes from APAPinjected mice constituted a discrete population with a high CD44 level ( Figure 1I). The subpopulation of hepatocytes also highly expressed PCNA and an HPC marker, 3 EpCAM ( Figure 1J,K, Figure S1B). To further identify the CD44expressing hepatocytes as HPCs, we verified their proliferation capacity. Considering the role of CD44 as an adhesion molecule that binds to hyaluronic acid (HA), we isolated small hepatocytes ( Figure S1C) and cultured them on an HA-coated dish. 4 The small hepatocytes isolated from APAP-injected mice proliferated and formed colonies ( Figure 1L). The cultured hepatocytes highly expressed CD44 with a low level of albumin, a representative marker for differentiated hepatocyte ( Figure S1D). Furthermore, AML12, a murine hepatocyte cell line, expressed a high level of CD44 and also proliferated on the HA-coated dish, indicating that AML12 cells could be a surrogate cell line for HPCs ( Figure 1M, Figure S1E).
To assess the role of CD44 in proliferative hepatocytes, we knocked down CD44 in AML12 ( Figure 2A). CD44 knockdown significantly enhanced tert-butyl hydrogen peroxide-induced reactive oxygen species (ROS) generation ( Figure 2B). Moreover, the intracellular glutathione (GSH) level was diminished by CD44 knockdown ( Figure 2C). Isotope tracing using universal 13 C 6 -cystine revealed that more than 90% of the intracellular GSH was derived from extracellular cystine (M3) in AML12 ( Figure  S2A). System x c − is a major antiporter that transports extracellular cystine for intracellular glutamate. 5 The expression of xCT was significantly decreased after knockdown of CD44 ( Figure 2D,E, Figure S2B,C). The activity of the system x c − antiporter was measured using an extracellular glutamate assay and universal 13 C 6 -cystine isotope tracing. Knockdown of CD44 hindered the activity of system x c − ( Figure 2F,G).
We established stable CD44-knockout AML12 ( Figure 2H). Immunoprecipitation analysis revealed that CD44 binds to xCT ( Figure 2I, Figure S2D). Sulforaphaneinduced xCT expression was diminished, but basal expression of xCT was not significantly changed after CD44 deletion ( Figure S2E). In fact, stable knockout of CD44 increased transcription of xCT (Slc7a11) and 4F2hc (Slc3a2) ( Figure S2F). We confirmed that nuclear factor erythroid 2-related factor 2, a major transcription factor of xCT, 6 was activated in the CD44-knockout AML12 ( Figure  S2G-I). We estimate that the oxidative stress-responsive signaling blunted the effect of CD44 on system x c − .
Meanwhile, CD44 ablation facilitated the protein degradation of xCT ( Figure 2J). The decreased protein stability  impeded formation of the 4F2hc/xCT complex ( Figure 2K). Deletion of CD44 significantly inhibited glutamate excretion ( Figure S2J) and decreased the intracellular levels of cysteine and GSH ( Figure 2L, Figure S2K). We also found that the limitations in cysteine and GSH resulted in a marked decrease in the proliferation rate ( Figure 2M). In addition to system x c − -mediated cystine uptake, cysteine can also be synthesized from methionine through the transsulfuration pathway composed of cystathionine β-synthase and cystathionine γ-lyase (CSE). 7 Expression of CSE was significantly increased after CD44 deletion. However, ablation of CD44 showed neither increase nor decrease of transsulfuration enzyme activity ( Figure  S2L,M). Primary hepatocytes were isolated from mice 48 h after APAP injection. Although the transcript levels of xCT did not change in the isolated primary hepatocytes ( Figure 3A), APAP injection up-regulated the expression of xCT in the plasma membrane ( Figure 3B). The activity of system x c − was markedly up-regulated by APAP injection ( Figure 3C-F, Figure S3A). The functional involvement of system x c − in proliferative hepatocytes was assessed using erastin, a selective inhibitor of system x c − . Although differentiated hepatocytes were resistant to erastin treatment, HPCs and AML12 were highly sensitive to it ( Figure 3G, Figure S3B). Overall, the data revealed that system x c − is necessary for HPC proliferation. We addressed the issue of whether system x c −dependent cystine uptake plays a critical role in liver regeneration. To that end, C57BL/6 mice were administered a metabolically stable system x c − inhibitor, imidazole ketone erastin 8 (IKE), 24 h immediately following APAP injection ( Figure 4A). IKE administration significantly abrogated the increase of proliferation markers in APAPinjected mice without increasing liver damage at later time points ( Figure 4B-D, Figure S4A-D). IKE administration retarded the increase of HPC markers ( Figure 4E). These results indicate that HPC-mediated cell proliferation was inhibited by blockade of system x c − .
The obtained results delineate the central role of CD44 in modulating liver-regenerative capacity. Specifically, CD44 contributes to the intracellular redox balance and cell proliferation by enhancing extracellular cystine uptake through system x c − .

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.