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Collagen I is a major component of extracellular matrix in human skin, and is also widely used in a variety of skin-care products. In this study, we investigated the modulatory roles of collagen I on human immortalized keratinocytes HaCaT, especially when cells were irradiated with UVB. Interestingly, the cells grown on plates coated by molecular collagen I, but not fibrillar collagen I, acquired certain resistance against UVB damages, as shown by increased survival and reduced apoptosis. The accumulation of dysfunctional mitochondria in UVB-treated cells was attenuated by molecular collagen I-coating. Interestingly, molecular collagen I rescued the loss of mitochondrial biogenesis in cells treated with UVB. Loss of PINK1/parkin-mediated mitophagy was dominant for the accumulation of dysfunctional mitochondria after UVB irradiation. Of note, cells cultured on molecular collagen I-precoated plates exhibited reserved mitophagy after UVB irradiation, as reflected by the enhanced protein level of PINK1/parkin, increased mitochondrial ubiquitin and the co-localization of lysosomes and mitochondria. Moreover, in UVB-treated cells, inhibiting mitophagy by Cyclosporin A, or by silencing PINK1 or parkin, disturbed the resolution of mitochondrial stress and reduced the protective effect of molecular collagen I, indicating that mitophagy is pivotal for the protection of collagen I against UVB damage in keratinocytes HaCaT. Collectively, this study reveals an unexpected protective role of collagen I, which facilitates mitophagy to rescue cells under UVB irradiation, providing a new direction for clinical application of collagen products., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ferroptosis, an iron-dependent cell death, is caused by lipid peroxidation. Noteworthily, accumulation of iron and lipid peroxidation are found in the proximity of the neuritic plaque, a hallmark of Alzheimer's disease (AD), but the relationship between ferroptosis and neuroinflammation in AD is unclear. Silibinin, extracted from the Silybum marianum, is possibly developed as an agent for AD treatment from its neuroprotective effect, but the effect of silibinin on sporadic AD that accounts for more than 95% of AD remains unclear. To determine whether silibinin alleviates the pathogenesis of sporadic AD and investigate the underlying mechanisms, STZ-treated HT22 murine hippocampal neurons and intracerebroventricular injection of streptozotocin (ICV-STZ) rats, a sporadic AD model, were used in this study. Results show that silibinin not only promotes survival of STZ-treated HT22 cells, but also ameliorates the cognitive impairment and anxiety/depression-like behavior of ICV-STZ rats. We here demonstrate that silibinin evidently inhibits the protein level of p53 as well as upregulates the protein level of cystine/glutamate antiporter SLC7A11 and ferroptosis inhibitor GPX4, but not p21, leading to the protection against STZ-induced ferroptotic damage. Immunofluorescent staining also shows that accumulation of lipid peroxidation induced by ferroptotic damage leads to increased fluorescence of 8-oxo-deoxyguanosine (8-OHDG), a maker of oxidized DNA. The oxidized DNA then leaks to the cytoplasm and upregulates the expression of the stimulator of interferon gene (STING), which triggers the production of IFN-β and other inflammatory cascades including NF-κB/TNFα and NLRP3/caspase 1/IL-1β. However, the treatment with silibinin blocks the above pathological changes. Moreover, in HT22 cells with/without STZ treatment, GPX4-knockdown increases the protein level of STING, indicating that the ferroptotic damage leads to the activation of STING signaling pathway. These results imply that silibinin exerts neuroprotective effect on an STZ-induced sporadic AD model by downregulating ferroptotic damage and thus the downstream STING-mediated neuroinflammation., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Type 2 diabetes (T2DM) is induced by the abundance of glucose and lipids, which causes glucolipotoxicity to the pancreatic β-cells. Silibinin is a natural flavonoid possessing the regulatory activity on insulin production and therapeutic activity in diabetic mice; however, its effect on glucolipotoxicity is not fully explained. This in vitro study investigates the effects of silibinin on palmitic acid (PA) and high glucose (HG)-induced cell loss and ferroptosis of rat insulinoma INS-1 cells. In the cells treated with PA and HG, expressions of glucose transporter 4 (Glut4) and carnitine acyltransferase I (CPT1) for β-oxidation of fatty acids are reduced. Mitochondria are the metabolic organelles for glucose and fatty acids. The mitochondrial membrane potential (MMP) and ATP production were decreased, while the ROS level was elevated in the cells treated with PA and HG, indicating an induction of mitochondrial disorder. Cell loss was partially rescued by ferroptosis inhibition, suggesting an involvement of ferroptosis in the cells treated with PA and HG. More importantly, the increases in total iron, lipid ROS, MDA and COX-2, and the decrease in ferroptosis inhibitory molecules GSH, GPX4 and FSP1 appeared in the cells treated with PA and HG, confirming the occurrence of ferroptosis. Moreover, PINK1/parkin-mediated mitophagy, a vital process for selective elimination of damaged mitochondria, was blocked. Interestingly, silibinin rescued the mitochondria, restricted the ferroptosis and restored the mitophagy. By using the pharmacological stimulator and inhibitor of mitophagy, and si-RNA transfection to silence PINK1 expression, silibinin's protective effect against ferroptosis caused by PA and HG treatment was found to depend on mitophagy. Collectively, our current study reveals the new mechanisms for the protection of silibinin against the injury of INS-1 cells treated with PA and HG, elucidates the participation of ferroptosis in glucolipotoxicity, highlighting the involvement of mitophagy in defense against ferroptotic cell death., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Inc.)

Ultraviolet B (UVB) irradiation causes skin inflammation and apoptosis. Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in skin damages, little is known about the roles of mitochondrial dynamics in these processes. UVB irradiation increases abnormal mitochondrial content but decreases mitochondrial volume in immortalized human keratinocyte HaCaT cells. UVB irradiation resulted in marked upregulation of mitochondrial fission protein dynamin-related protein 1 (DRP1) and downregulation of mitochondrial outer membrane fusion proteins 1 and 2 (MFN1 and MFN2) in HaCaT cells. Mitochondrial dynamics was discovered to be crucial for NLRP3 inflammasome and cGAS-STING pathway activation, as well as the induction of apoptosis. Inhibition of mitochondrial fission by treatments with a DRP1 inhibitor, mdivi-1, or with DRP1-targeted siRNA, efficiently prevented UVB-induced NLRP3/cGAS-STING mediated pro-inflammatory pathways or apoptosis in the HaCaT cells, whereas inhibition of mitochondrial fusion with MFN1and 2 siRNA increased these pro-inflammatory pathways or apoptosis. The enhanced mitochondrial fission and reduced fusion caused the up-regulation of reactive oxygen species (ROS). Application of an antioxidant, N-acetyl-l-cysteine (NAC), which scavenges excessive ROS, attenuated inflammatory responses through suppressing NLRP3 inflammasome and cGAS-STING pathway activation, and rescued cells from apoptosis caused by UVB-irradiation. Together, our findings revealed the regulation of NLRP3/cGAS-STING inflammatory pathways and apoptosis by mitochondrial fission/fusion dynamics in UVB-irradiated HaCaT cells, providing a new strategy for the therapy of UVB skin injury., (Copyright © 2023. Published by Elsevier Inc.)

Ultraviolet B (UVB) irradiation causes skin damages. In this study, we focus on the involvement of mitochondrial disorders in UVB injury. Surprisingly, UVB irradiation increases the amounts of mitochondria in human immortalized keratinocytes HaCaT. However, further analysis shows that ATP levels decreased by UVB treatment in accordance with the collapse of mitochondrial membrane potential (MMP), suggesting an accumulation of dysfunctional mitochondria in UVB-irradiated HaCaT cells. Mitophagy, mainly mediated by PINK1 and parkin, is critical for the elimination of damaged mitochondria. Western blot results show that the levels of both PINK1 and parkin are decreased in UVB-irradiated cells, indicating the impairment of mitophagy. Silencing the expression of PINK1 or parkin by transfection of siRNA shows essentially the same damage to the cells as UVB irradiation does, including increased mitochondrial amount, decreased MMP and ATP production, and enhanced apoptosis, evidencing that repression of PINK1/parkin-mediated mitophagy plays a primary cause of UVB-caused cells damages. We previously found that HaCaT cells exposed to UVB showed activation of the cGAS-STING pathway and apoptosis. Here, silencing PINK1 or parkin also increases the protein levels of cGAS and STING, facilitates nuclear accumulation of NF-κB, and promotes the transcription of IFNβ, suggesting for the activation of STING pathway. Mitophagy impairment either by UVB-irradiation or by PINK1/parkin silencing initiates caspase-3-mediated apoptosis, as shown by the activation of caspase-3 and cleavage of PARP, as well as the increase of Hoechst-positive stained cells and Annexin V-positive cells. Further studies find that Bax-mediated permeabilization of mitochondrial membrane is critical for cell apoptosis, as well as the cytosolic leakage of mtDNA in UVB-treated cells, which results in cGAS-STING activation, and these processes are negatively-regulated by PINK1/parkin-mediated mitophagy. This study reveals the involvement of dysfunctional mitochondria due to impaired mitophagy in the damaging effect of UVB irradiation on HaCaT cells. Restoring the mitophagy has the potential to be developed as a new strategy to protect skin from UVB damages., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The extracellular matrix (ECM) regulates cell behavior through signal transduction and provides a suitable place for cell survival. As one of the major components of the extracellular matrix, type I collagen is involved in regulating cell migration, proliferation and differentiation. We present a system in which 3T3-L1 preadipocyte cells are induced for adipogenic differentiation on type I collagen coated dishes. Our previous study has found that type I collagen inhibits adipogenic differentiation via YAP activation. Here we further reveal that type I collagen inactivates autophagy by up-regulating mTOR activity via the YAP pathway. Under collagen-coating conditions, co-localization of lysosomes with mTOR was increased and the level of downstream protein p-S6K was elevated, accompanied by a decrease in the level of autophagy. Autophagy is negatively correlated with adipogenesis under type I collagen coating. Through the YAP-autophagy axis, type I collagen improves glycolipid metabolism accompanied by increased mitochondrial content, enhanced glucose uptake, reduced release of free fatty acids (FFAs) and decreased intracellular lipid accumulation. Our findings provide insight into the strategy for dealing with obesity: Type I collagen or the drugs with inhibitory effects on autophagy or YAP, have a potential to accelerate the energy metabolism of adipose tissue, so as to better maintain the homeostasis of glucose and lipids in the body, which can be used for achieving weight loss., (Copyright © 2022. Published by Elsevier B.V.)

We reported previously that higher doses (150-250 μM) of silibinin enhanced fission and inhibited fusion of mitochondria, accompanying apoptosis of double-positive breast cancer cell line MCF-7 cells and triple-negative breast cancer cell line MDA-MB-231 cells. We report here three important questions yet unclarified in the previous study; 1) Whether enhanced fission of mitochondria by the treatment of silibinin leads to mitophagy, 2) Whether mitophagy positively contributes to apoptosis and 3) Whether estrogen receptor-positive (ER + ) MCF-7 cells and estrogen receptor-negative (ER - ) MDA-MB-231 cells are affected in a different way by silibinin treatment, since silibinin often works through ERs signaling pathway. Mitophagy driven by Pink1/Parkin signaling, plays an important role in eliminating damaged mitochondria. Indeed, increased expression of Pink1 and the recruitment of Parkin and LC3-II to mitochondria by the treatment with silibinin account for silibinin induction of mitophagy. In this study, the effects of mitochondrial division inhibitor 1 (mdivi-1) and small interfering RNA targeting dynamin-related protein 1 (DRP1) were examined to reveal the effect of mitochondrial fission on mitophagy. As expected, mdivi-1 or siRNA targeting DRP1 reversed silibinin-induced mitochondrial fission due to down-regulation in the expression of DRP1. Inhibition of mitochondrial fission by mdivi-1 prevented induction of mitophagy as well as autophagy in both MCF-7 and MDA-MB-231 cells, indicating that silibinin-induced mitochondrial fission leads to mitophagy. Inhibition of mitochondrial fission efficiently prevented silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells in our previous work, and the second point of the present study, inhibition of mitophagy by Pink1 or Parkin knockdown increased silibinin-induced apoptosis of these cells, respectively, suggesting that the mitophagy induced by silibinin treatment serves as a cytoprotective effect, resulting in reduction of apoptosis of cancer cells in both cells. In the third point, we studied whether estrogen receptors (ERs) played a role in silibinin-induced mitophagy and apoptosis in MCF-7 and MDA-MB-231 cells. ERα and ERβ are not involved in silibinin-induced mitophagic process in MCF-7 and MDA-MB-231 cells. These findings demonstrated that silibinin induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis not through ERs-Pink1 or -Parkin pathway in MCF-7 and MDA-MB-231., (Copyright © 2020. Published by Elsevier Inc.)

Mitochondria are dynamically regulated by fission and fusion processes. Silibinin induces apoptosis of MCF-7 and MDA-MB-231 human breast cancer cells. However, whether or not mitochondria dysfunction is involved in the apoptosis induction with silibinin of both types of the cells remains unknown. We here report that silibinin decreases the mitochondrial mass in terms of MitoTracker Green staining in both breast cancer cells. Silibinin induces morphological changes of mitochondria from oval to truncated or fragmented shapes accordingly. Condensed crests are observed in mitochondria by transmission electron microscopy. Silibinin causes mitochondrial membrane potential reduced. The expression of mitochondrial fission-associated proteins including dynamin-related protein 1 (DRP1) is up-regulated, whereas expression of the mitochondrial fusion-associated proteins, optic atrophy 1 and mitofusin 1, is down-regulated. In addition, silibinin treatment down-regulates ATP content as well as the levels of mitochondrial biogenesis-regulators including mitochondrial transcription factor A, peroxisome proliferator-activated receptor gamma coactivator 1 and nuclear respiratory factor 2. Moreover, treatments with DRP1 inhibitor, mdivi-1, or with DRP1-targetted siRNA efficiently prevent silibinin-induced apoptosis in the breast cancer cells, whereas inhibition of DRP1 phosphorylation with staurosporine increases apoptosis furthermore. Taken together, we conclude that silibinin impairs mitochondrial dynamics and biogenesis, leading to apoptosis of MCF-7 and MDA-MB-123 cells., (Copyright © 2019. Published by Elsevier Inc.)

Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We recognized for the first time upregulation of type VIII collagen gene expression during the repair process in the mouse brain cold injury model. Immunohistochemical staining showed that type VIII collagen expression was around the necrotic region, where reactive astrocytes are frequently observed. Cultured astrocytes demonstrated a high expression of type VIII collagen genes. TGF-beta1 enhanced the expression of both alpha1(VIII) and alpha2(VIII) genes by astrocytes in culture. Further, we tested selected biological activities of type VIII collagen, compared with those of type I, IV, and V collagens and fibronectin. Astrocytes adhered to type VIII collagen via receptors requiring metal ions. Astrocyte migration on type VIII collagen was more stimulated than that observed on the other ECM molecules. These data indicate that type VIII collagen plays an important role in glial scar formation during the repair process by astrocytes.

To investigate the pathophysiologic role of matrix metalloproteinase 9 (MMP-9), we analyzed the mechanism of its transcriptional regulation in keratinocytes and in HT1080 fibrosarcoma cells in culture. The KRE-M9 element, which is located between the 12-O-tetradecanoyl-phorbol-13-acetate responsive element (TRE) and the transcription initiation site in the MMP-9 promoter, is essential for MMP-9 transcription in the absence of the TRE. The KRE-M9 binding protein, however, is shown to be a repressor of transcription rather than an activator; we found several times higher transcriptional activity when the KRE-M9 element was mutated. In contrast, activator protein 1 proteins (AP-1) are shown to activate transcription of MMP-9 by binding to the TRE, which is located adjacent to the KRE-M9 element. Moreover, we found that the KRE-M9 binding protein could serve as a differentiation repressing factor 1 (DRF-1) as shown by the decrease in levels of this protein with differentiation. In addition, the TRE binding protein is able to bind to the KRE-M9 to some extent. These results indicate that the coordinated modulation of MMP-9 transcription via the TRE and the KRE-M9 elements is important in epidermal and mesenchymal tissues. Our findings could facilitate consideration of the molecular mechanism in a variety of pathophysiologic conditions with which MMP-9 is involved.

Our previous reports showed that cultured human cells secrete non-disulfide-bonded non-helical alpha1(IV) and alpha2(IV) chains under physiological conditions. In the present report we show that the alpha(IV) chains in non-helical form were reactive to lectin ABA (Agaricus bisporus agglutinin), whereas the alpha(IV) chains secreted in triple-helical form were not. These results indicate that ABA could be used to distinguish the two conformational isomers of type IV collagen polypeptides. An alpha1(IV) chain isolated from human placenta with an antibody-coupled column showed a positive reaction to ABA, indicating that gelatin form of the type IV collagen alpha1(IV) chain is produced and retained in the tissue in vivo. A possible significance of the gelatin form is discussed from the finding that the non-helical alpha1(IV) chain purified with EDTA-free buffer contained degraded polypeptides including NC1-size domain and showed an apparent inhibition against activated pro-MMP-9. This is the first report to show that a gelatin form of protein exists in vivo.

Intranuclear inclusions have been observed in the brains of patients affected with Huntington's disease (HD). Neuro 2A cells that transiently expressed HD exon 1 bearing 74 glutamine repeats linked to the green fluorescent protein (GFP) and the nuclear localization sequence (NLS) contained aggregates in nuclei. The aggregates were purified by fractionation with centrifugation followed by fluorescence-activated cell sorting (FACS). Heat treatment of the aggregate in an SDS sample buffer caused the dense aggregate cores to disappear and generated a basket-like structure composed of fibrils. Biochemical analysis of the aggregates revealed that the HD exon 1-GFP fusion protein was the major component. The heterogeneous nuclear ribonucleoproteins F and H, histones and ubiquitin were found to be associated with the aggregates. Our observations suggest that the N-terminal fragment of huntingtin may organize the skeletal structure of the aggregates and may disturb normal cellular functions by trapping other proteins within the aggregates., ((c) 2002 Elsevier Science (USA).)

Zymography and reverse zymography are widely used techniques for identifying the proteolytic activity of enzymes and the presence of protease inhibitors in polyacrylamide gels. In the current studies, we utilized a fluorescein-isothiocyanate-labeled substrate to develop novel zymographic and reverse zymographic methods for detecting matrix metalloproteinases and tissue inhibitors of the metalloproteinases, respectively. Using a transilluminator, the results can be observed visually without stopping the enzymatic reaction. For this reason, we have named these methods real-time zymography and real-time reverse zymography. These methods have the following advantages compared with conventional protocols: (1) because the reaction can be repeatedly monitored on the polyacrylamide gels, optimization of the incubation time can be achieved without preliminary analyses; (2) higher sensitivity is achieved with a lower amount of substrate than with conventional methods; (3) a semi-quantitative analysis of matrix metalloproteinases is possible. An additional advantage of the real-time reverse zymography is that, because the fluorescence detection is specific for substrate digestion, the inhibitor bands can be easily distinguished from contaminating proteins.