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Background <p>CD36, a vital fatty acid translocase, has been reported to participate in multiple physiological functions through palmitoylation mediated by zinc finger Asp–His–His–Cys-type palmitoyltransferases (DHHCs). This study aimed to investigate the possible involvement of DHHC-mediated CD36 palmitoylation in high-fat diet (HFD)-induced impairment of pubertal mammary gland development and explore the underlying mechanisms involved.</p> Methods <p>Palmitic acid (PA)-treated HC11 cells were used as the in&#xa0;vitro high-fat model, and the cell proliferation was examined by 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay. The palmitoylation of CD36 was determined by the acyl–biotin exchange (ABE) method. The expression of CD36, proliferative genes, and signaling molecules was detected by immunoblotting. The cellular localization of CD36 was determined by immunofluorescence. The bindings of CD36 with zinc finger DHHC-type palmitoyltransferases 9 (DHHC9) or Fyn/Lyn were detected by co-immunoprecipitation (Co-IP). The palmitoylation inhibitor 2-bromopalmitate (2BP), DHHC9 knockdown, and point mutation of CD36 cysteine residues were applied to construct a CD36 palmitoylation deficiency model in&#xa0;vitro to investigate the effects of CD36 palmitoylation on HC11 proliferation. In vivo, the pubertal mice were treated with HFD and/or 2BP. Mammary gland morphology was determined by whole mount staining, and the underlying mechanisms were verified by the methods used in the in vitro system.</p> Results <p>In vitro, the palmitoylation inhibitor 2BP eliminated PA-inhibited HC11 proliferation and inhibited CD36 palmitoylation and localization on the plasma membrane. Meanwhile, the binding of DHHC9 and CD36 in PA-treated HC11 cells was repressed by 2BP. In addition, both knockdown of DHHC9 and point mutation of CD36 cysteine residues suppressed the membrane palmitoylation and localization of CD36 and stimulated the proliferation of PA-treated HC11 cells. Furthermore, in PA-treated HC11 cells, the inhibition of CD36 palmitoylation, the knockdown of DHHC9, and the mutation of CD36 cysteine residues resulted in decreased formation of the CD36/Fyn/Lyn complex. Correspondingly, the downstream c-jun n-terminal kinase 1 (JNK1) pathway was inhibited, and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway was activated. Moreover, inhibition of the JNK pathway with SP600125 promoted the proliferation of PA-treated HC11 cells via activation of the ERK1/2 pathway. In vivo, the palmitoylation inhibitor 2BP ameliorated HFD-induced impairment of mammary gland development in pubertal female mice, which was associated with a decrease in DHHC9-mediated CD36 palmitoylation in the plasma membrane, a reduction in the CD36/Fyn/Lyn complex, inhibition of the JNK1 pathway, and activation of the ERK1/2 pathway.</p> Conclusions <p>This study revealed that inhibition of DHHC9-mediated CD36 palmitoylation mitigated HFD-induced impairment of pubertal mammary gland development via the JNK1-ERK1/2 pathway.</p> Graphical Abstract <p></p>

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Inhibition of DHHC9-mediated CD36 palmitoylation lessens high-fat diet (HFD)-induced impairment of pubertal mammary gland development through the JNK-ERK pathway

  • 期刊论文

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  • 发表日期：

    2025年10月14日

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  • Yiming Fu,
  • Fenglin Zhang,
  • Limin Lang,
  • Shuyi Liang,
  • Junfeng Wang,
  • Shilei Zhang,
  • Kaixin Deng,
  • Jinhao Liu,
  • Lilin Cai,
  • Yue Zhang,
  • Lidong Yan,
  • Guo Ren,
  • Haoyan Kang,
  • Lina Wang,
  • Canjun Zhu,
  • Ruifan Wu,
  • Gang Shu,
  • Qingyan Jiang,
  • Songbo Wang

摘要

Background

CD36, a vital fatty acid translocase, has been reported to participate in multiple physiological functions through palmitoylation mediated by zinc finger Asp–His–His–Cys-type palmitoyltransferases (DHHCs). This study aimed to investigate the possible involvement of DHHC-mediated CD36 palmitoylation in high-fat diet (HFD)-induced impairment of pubertal mammary gland development and explore the underlying mechanisms involved.

Methods

Palmitic acid (PA)-treated HC11 cells were used as the in vitro high-fat model, and the cell proliferation was examined by 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay. The palmitoylation of CD36 was determined by the acyl–biotin exchange (ABE) method. The expression of CD36, proliferative genes, and signaling molecules was detected by immunoblotting. The cellular localization of CD36 was determined by immunofluorescence. The bindings of CD36 with zinc finger DHHC-type palmitoyltransferases 9 (DHHC9) or Fyn/Lyn were detected by co-immunoprecipitation (Co-IP). The palmitoylation inhibitor 2-bromopalmitate (2BP), DHHC9 knockdown, and point mutation of CD36 cysteine residues were applied to construct a CD36 palmitoylation deficiency model in vitro to investigate the effects of CD36 palmitoylation on HC11 proliferation. In vivo, the pubertal mice were treated with HFD and/or 2BP. Mammary gland morphology was determined by whole mount staining, and the underlying mechanisms were verified by the methods used in the in vitro system.

Results

In vitro, the palmitoylation inhibitor 2BP eliminated PA-inhibited HC11 proliferation and inhibited CD36 palmitoylation and localization on the plasma membrane. Meanwhile, the binding of DHHC9 and CD36 in PA-treated HC11 cells was repressed by 2BP. In addition, both knockdown of DHHC9 and point mutation of CD36 cysteine residues suppressed the membrane palmitoylation and localization of CD36 and stimulated the proliferation of PA-treated HC11 cells. Furthermore, in PA-treated HC11 cells, the inhibition of CD36 palmitoylation, the knockdown of DHHC9, and the mutation of CD36 cysteine residues resulted in decreased formation of the CD36/Fyn/Lyn complex. Correspondingly, the downstream c-jun n-terminal kinase 1 (JNK1) pathway was inhibited, and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway was activated. Moreover, inhibition of the JNK pathway with SP600125 promoted the proliferation of PA-treated HC11 cells via activation of the ERK1/2 pathway. In vivo, the palmitoylation inhibitor 2BP ameliorated HFD-induced impairment of mammary gland development in pubertal female mice, which was associated with a decrease in DHHC9-mediated CD36 palmitoylation in the plasma membrane, a reduction in the CD36/Fyn/Lyn complex, inhibition of the JNK1 pathway, and activation of the ERK1/2 pathway.

Conclusions

This study revealed that inhibition of DHHC9-mediated CD36 palmitoylation mitigated HFD-induced impairment of pubertal mammary gland development via the JNK1-ERK1/2 pathway.

Graphical Abstract