Abstract
Background: Autophagy is deeply associated with aging, but little is known about its association with the extracellular matrix (ECM). 3-methyladenine (3-MA) is a commonly used autophagy inhibitor.
Objective: We used this compound to investigate the role of autophagy in dermal ECM protein synthesis.
Methods: Normal human dermal fibroblasts (NHDFs) were treated with 3-MA for 24 h, and mRNA encoding several ECM proteins was analyzed in addition to the protein expression of procollagen-1 and fibronectin. Several phosphoinositide 3-kinase (PI3K) inhibitors, an additional autophagy inhibitor, and small interfering RNA (siRNA) targeting autophagy-related genes were additionally used to confirm the role of autophagy in ECM synthesis.
Results: Only 3-MA, but not other chemical compounds or autophagy-related genetargeting siRNA, inhibited the transcription of procollagen-1 and fibronectin-encoding genes. Further, 3-MA did not affect the activation of regulatory Smads, but inhibited the interaction between Smad3 with p300. Moreover, 3-MA treatment increased the phosphorylation of cAMP response element-binding protein (CREB); however, CREB knock-down did not recover 3-MA-induced procollagen-1 and fibronectin downregulation.
Conclusion: We revealed that 3-MA might inhibit procollagen-1 and fibronectin synthesis in an autophagy-independent manner by interfering with the binding between Smad3 and p300. Therefore, 3-MA could be a candidate for the treatment of diseases associated with the accumulation of ECM proteins.
Keywords: 3-methyladenine, autophagy, CREB, dermal extracellular matrix, normal human dermal fibroblasts, Smad3–p300 binding.
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