Abstract:The expression and processing of laggrin, a lament-associated protein in the skin epidermis, is closely associated with keratinocyte corni cation. The large precursor pro laggrin (Pro-FLG) is initially detected at the granular layer in keratohyalin granules, subsequently processed into ten to twelve laggrin monomers (mFLGs) for keratin assembly, and ultimately degraded into smaller peptides that behave as natural moisturizing factor (NMF) at the outermost epidermis. We previously reported that a t-SNARE protei… Show more
“…1 ). Furthermore, it has been reported that FLG is very unstable in HaCaT cells, which express caspase14 to digest FLG into small peptides known as natural moisturizing factors (NMFs) 12 . Figure 2 The regulation of mesotrypsin expression in HaCaT cells is governed by a protein elimination system.…”
Section: Resultsmentioning
confidence: 99%
“…Analysis of the samples was conducted using a TCS SPE system (Leica, Wetzlar, Germany). The primary antibodies utilized in this study included antibodies against β-actin (Sigma‒Aldrich), TGase-1 (Proteintech, Rosemont, USA), E-cadherin (ECCD2, a gift from Dr. Takeichi), LaminA/C (Cell Signaling, Danvers, USA), Lamin B1 (Abcam, Cambridge, UK), Rb, phosphorylated Rb (Cell Signaling Technology, Danvers, USA), ZO-1 (a gift from Dr. Nagafuchi), T7-tag (MBL, Tokyo, Japan), Ki67 (Abcam), Pro-FLG 12 , and GFP/Venus (culture supernatant of JFP-J1 cells, Riken Cell Bank RCB2309). Secondary antibodies conjugated to Alexa488, Cy3, or horseradish peroxidase (HRP) were obtained from Sigma‒Aldrich and Merck-Millipore (Darmstadt, Germany).…”
Section: Methodsmentioning
confidence: 99%
“…Initially, expressed as a large phosphorylated precursor, pro-filaggrin (Pro-FLG), in the granular layers exists as nonfunctional keratohyalin granules. Pro-FLG, comprising an N-terminal calcium-binding domain (FLG-N) and multiple filaggrin units (FLG), undergoes rapid processing and release by various proteases, including kallikrein5 (KLK5), KLK7, calpain, RACE4, SASPase, and mesotrypsin 12 – 14 . FLG-N activates the cell death program in granular cell nuclei, while FLG not only bundles keratin intermediate filaments but also influences actin dynamics, controlling cell shape and cell–cell adhesion 2 , 12 , 15 , 16 .…”
Section: Introductionmentioning
confidence: 99%
“…Pro-FLG, comprising an N-terminal calcium-binding domain (FLG-N) and multiple filaggrin units (FLG), undergoes rapid processing and release by various proteases, including kallikrein5 (KLK5), KLK7, calpain, RACE4, SASPase, and mesotrypsin 12 – 14 . FLG-N activates the cell death program in granular cell nuclei, while FLG not only bundles keratin intermediate filaments but also influences actin dynamics, controlling cell shape and cell–cell adhesion 2 , 12 , 15 , 16 . Additionally, FLG acts as a component of cornified cell envelopes, robust structures formed beneath the cell membrane 15 , 17 .…”
Section: Introductionmentioning
confidence: 99%
“…However, unlike normal keratinocytes, HaCaT cells can proliferate in differentiated states without manifesting a flattened cell morphology 26 , 28 . Recent observations indicate that these cells maintained in conventional high-calcium medium express significant amounts of Pro-FLG irrespective of differentiation state and exhibit insufficient Pro-FLG processing systems 12 . Building upon these results, we sought to elucidate the role of mesotrypsin in keratinocytes utilizing HaCaT cells harboring a tetracycline (tet)/doxycycline (dox)-inducible expression cassette for active mesotrypsin.…”
Mesotrypsin, encoded by the PRSS3 gene, is a distinctive trypsin isoform renowned for its exceptional resistance to traditional trypsin inhibitors and unique substrate specificity. Within the skin epidermis, this protein primarily expresses in the upper layers of the stratified epidermis and plays a crucial role in processing pro-filaggrin (Pro-FLG). Although prior studies have partially elucidated its functions using primary cultured keratinocytes, challenges persist due to these cells' differentiation-activated cell death program. In the present study, HaCaT keratinocytes, characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states, were utilized to further scrutinize the function of mesotrypsin. Despite the ready degradation of the intact form of active mesotrypsin in these cells, fusion with Venus, flanked by a peptide linker, enables evasion from the protein elimination machinery, thus facilitating activation of the Pro-FLG processing system. Inducing Venus-mesotrypsin expression in the cells resulted in a flattened phenotype and reduced proliferative capacity. Moreover, these cells displayed altered F-actin assembly, enhanced E-cadherin adhesive activity, and facilitated tight junction formation without overtly influencing epidermal differentiation. These findings underscore mesotrypsin's potentially pivotal role in shaping the characteristic cellular morphology of upper epidermal layers.
“…1 ). Furthermore, it has been reported that FLG is very unstable in HaCaT cells, which express caspase14 to digest FLG into small peptides known as natural moisturizing factors (NMFs) 12 . Figure 2 The regulation of mesotrypsin expression in HaCaT cells is governed by a protein elimination system.…”
Section: Resultsmentioning
confidence: 99%
“…Analysis of the samples was conducted using a TCS SPE system (Leica, Wetzlar, Germany). The primary antibodies utilized in this study included antibodies against β-actin (Sigma‒Aldrich), TGase-1 (Proteintech, Rosemont, USA), E-cadherin (ECCD2, a gift from Dr. Takeichi), LaminA/C (Cell Signaling, Danvers, USA), Lamin B1 (Abcam, Cambridge, UK), Rb, phosphorylated Rb (Cell Signaling Technology, Danvers, USA), ZO-1 (a gift from Dr. Nagafuchi), T7-tag (MBL, Tokyo, Japan), Ki67 (Abcam), Pro-FLG 12 , and GFP/Venus (culture supernatant of JFP-J1 cells, Riken Cell Bank RCB2309). Secondary antibodies conjugated to Alexa488, Cy3, or horseradish peroxidase (HRP) were obtained from Sigma‒Aldrich and Merck-Millipore (Darmstadt, Germany).…”
Section: Methodsmentioning
confidence: 99%
“…Initially, expressed as a large phosphorylated precursor, pro-filaggrin (Pro-FLG), in the granular layers exists as nonfunctional keratohyalin granules. Pro-FLG, comprising an N-terminal calcium-binding domain (FLG-N) and multiple filaggrin units (FLG), undergoes rapid processing and release by various proteases, including kallikrein5 (KLK5), KLK7, calpain, RACE4, SASPase, and mesotrypsin 12 – 14 . FLG-N activates the cell death program in granular cell nuclei, while FLG not only bundles keratin intermediate filaments but also influences actin dynamics, controlling cell shape and cell–cell adhesion 2 , 12 , 15 , 16 .…”
Section: Introductionmentioning
confidence: 99%
“…Pro-FLG, comprising an N-terminal calcium-binding domain (FLG-N) and multiple filaggrin units (FLG), undergoes rapid processing and release by various proteases, including kallikrein5 (KLK5), KLK7, calpain, RACE4, SASPase, and mesotrypsin 12 – 14 . FLG-N activates the cell death program in granular cell nuclei, while FLG not only bundles keratin intermediate filaments but also influences actin dynamics, controlling cell shape and cell–cell adhesion 2 , 12 , 15 , 16 . Additionally, FLG acts as a component of cornified cell envelopes, robust structures formed beneath the cell membrane 15 , 17 .…”
Section: Introductionmentioning
confidence: 99%
“…However, unlike normal keratinocytes, HaCaT cells can proliferate in differentiated states without manifesting a flattened cell morphology 26 , 28 . Recent observations indicate that these cells maintained in conventional high-calcium medium express significant amounts of Pro-FLG irrespective of differentiation state and exhibit insufficient Pro-FLG processing systems 12 . Building upon these results, we sought to elucidate the role of mesotrypsin in keratinocytes utilizing HaCaT cells harboring a tetracycline (tet)/doxycycline (dox)-inducible expression cassette for active mesotrypsin.…”
Mesotrypsin, encoded by the PRSS3 gene, is a distinctive trypsin isoform renowned for its exceptional resistance to traditional trypsin inhibitors and unique substrate specificity. Within the skin epidermis, this protein primarily expresses in the upper layers of the stratified epidermis and plays a crucial role in processing pro-filaggrin (Pro-FLG). Although prior studies have partially elucidated its functions using primary cultured keratinocytes, challenges persist due to these cells' differentiation-activated cell death program. In the present study, HaCaT keratinocytes, characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states, were utilized to further scrutinize the function of mesotrypsin. Despite the ready degradation of the intact form of active mesotrypsin in these cells, fusion with Venus, flanked by a peptide linker, enables evasion from the protein elimination machinery, thus facilitating activation of the Pro-FLG processing system. Inducing Venus-mesotrypsin expression in the cells resulted in a flattened phenotype and reduced proliferative capacity. Moreover, these cells displayed altered F-actin assembly, enhanced E-cadherin adhesive activity, and facilitated tight junction formation without overtly influencing epidermal differentiation. These findings underscore mesotrypsin's potentially pivotal role in shaping the characteristic cellular morphology of upper epidermal layers.
Iron is a vital metal for most biological functions in tissues, and its concentration is exquisitely regulated at the cellular level. During the process of differentiation, keratinocytes in the epidermis undergo a noticeable reduction in iron content. Conversely, psoriatic lesions, characterized by disruptions in epidermal differentiation, frequently reveal an excessive accumulation of iron within keratinocytes that have undergone differentiation. In this study, we clarified the significance of attenuated cellular iron content in the intricate course of epidermal differentiation. We illustrated this phenomenon through the utilization of hinokitiol, an iron chelator derived from the heartwood of Taiwanese hinoki, which forcibly delivers iron into cells independent of the intrinsic iron-regulation systems. While primary cultured keratinocytes readily succumbed to necrotic cell death by this iron chelator, mild administration of the hinokitiol-iron complex modestly disrupts the process of differentiation in these cells. Notably, keratinocyte model cells HaCaT and anaplastic skin rudiments exhibit remarkable resilience against the cytotoxic impact of hinokitiol, and the potent artificial influx of iron explains a suppressive effect selectively on epidermal differentiation. Moreover, the augmentation of iron content induced by the overexpression of divalent metal transporter 1 (DMT1) culminates in the inhibition of differentiation in HaCaT cells. Consequently, the diminution in cellular iron content emerges as an important determinant influencing the trajectory of keratinocyte differentiation.
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