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Calpain inhibitors reduce the cornified cell envelope formation by inhibiting proteolytic processing of
Abstract:
Calpain I (mu-calpain) and II (m-calpain) are well known calcium-activated neutral cysteine proteases. Many reports have shown that activation of calpain is related to cataract formation, neuronal degeneration, blood clotting, ischemic injuries, muscular dystrophy and cornified cell envelope (CE) formation. Here, we report that insoluble CE formation was reduced after treatment with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal) on normal human epidermal keratinocytes (NHEK), whereas serine and thiol protease inhibitors had no effect on the reduction of CE. When NHEK cells were confluent, keratinocytes were treated with various concentrations (0.5 microM-0.5 mM) of calpain I inhibitor or serine and thiol protease inhibitors under calcium induced differentiation. Insoluble CE formation was reduced about 90% in the 50 microM calpain inhibitor I treated group by day 9 of culture, whereas insoluble CE was reduced only 10% in the same condition. Interestingly TGase activity was blocked by 90% in the 0.5 mM calpain inhibitor treated group within 72 h, whereas TGase activity was retained by 80% in the 0.5 mM serine protease inhibitor treated group at 7 day treatment. Therefore it can be suggested that cysteine protease calpains might be responsible for the activation of the TGase 1 enzyme to complete insoluble CE formation during epidermal differentiation.
Insights
Calpain inhibitors significantly reduced cornified envelope formation in human keratinocytes, suggesting calpains activate TGase 1 for epidermal differentiation. This finding impacts understanding of skin barrier development.
Area of Science:
- Biochemistry
- Dermatology
- Cell Biology
Background:
- Calpains are calcium-activated cysteine proteases implicated in various biological processes.
- Calpain activation is linked to conditions like cataract formation and neuronal degeneration.
- Their role in epidermal differentiation and cornified envelope formation requires further elucidation.
Purpose of the Study:
- To investigate the role of calpains in the formation of insoluble cornified envelopes (CE) during epidermal differentiation.
- To determine if calpain inhibition affects TGase 1 activity, crucial for CE formation.
Main Methods:
- Normal human epidermal keratinocytes (NHEK) were treated with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal).
- Cells were cultured under calcium-induced differentiation conditions with varying inhibitor concentrations.
- Insoluble CE formation and TGase activity were measured over time.
Main Results:
- Calpain I inhibition (50 microM) reduced insoluble CE formation by approximately 90% by day 9.
- TGase activity was blocked by 90% within 72 hours with 0.5 mM calpain inhibitor treatment.
- Serine and thiol protease inhibitors showed minimal effect on CE formation and TGase activity.
Conclusions:
- Cysteine protease calpains are likely responsible for activating TGase 1.
- This activation is essential for the complete formation of insoluble CE during epidermal differentiation.
- Calpains represent a potential target for modulating skin barrier function.