Assessment of Tilapia Skin Collagen for Biomedical Research Applications in Comparison with Mammalian Collagen.

Collagen is an important material for biomedical research, but using mammalian tissue-derived collagen carries the risk of zoonotic disease transmission. Marine organisms, such as farmed tilapia, have emerged as a safe alternative source of collagen for biomedical research. However, the tilapia collagen products for biomedical research are rare, and their biological functions remain largely unexamined. In this study, we characterized a commercial tilapia skin collagen using SDS-PAGE and fibril formation assays and evaluated its effects on skin fibroblast adhesion, proliferation, and migration, comparing it with commercial collagen from rat tails, porcine skin, and bovine skin. The results showed that tilapia skin collagen is a type I collagen, similar to rat tail collagen, and has a faster fibril formation rate and better-promoting effects on cell migration than porcine and bovine skin collagen. We also confirmed its application in a 3D culture for kidney cells' spherical cyst formation, fibroblast-induced gel contraction, and tumor spheroid interfacial invasion. Furthermore, we demonstrated that the freeze-dried tilapia skin collagen scaffold improved wound closure in a mouse excisional wound model, similar to commercial porcine or bovine collagen wound dressings. In conclusion, tilapia skin collagen is an ideal biomaterial for biomedical research.

Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12 -induced cell transformation.

Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-RasV12 gene) transformation by Ha-RasV12 . Cav1 overexpression abrogates the Ha-RasV12 -driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-RasV12 -inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-RasV12 , was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-RasV12 - and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-RasV12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-RasV12 -Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-RasV12 -driven cell transformation.

Overexpression of exogenous kidney-specific Ngal attenuates progressive cyst development and prolongs lifespan in a murine model of polycystic kidney disease.

Neutrophil gelatinase-associated lipocalin (Ngal) is a biomarker for acute and chronic renal injuries, including polycystic kidney disease (PKD). However, the effect of Ngal on PKD progression remains unexplored. To study this, we generated 3 strains of mice with different expression levels of Ngal within an established PKD model (Pkd1L3/L3): Pkd1L3/L3 (with endogenous Ngal), Pkd1L3/L3; NgalTg/Tg (with endogenous and overexpression of exogenous kidney-specific Ngal) and Pkd1L3/L3; Ngal-/- mice (with Ngal deficiency). Knockout of endogenous Ngal had no effect on phenotypes, cystic progression, or survival of the PKD mice. However, the transgenic mice had a significantly longer lifespan, smaller (but not fewer) renal cysts, and less interstitial fibrosis than the mice without or with endogenous Ngal. Western-blot analyses showed significant increases in Ngal and cleaved caspase-3 and decreases in α-smooth muscle actin, hypoxia-inducible factor 1-α, pro-caspase 3, proliferating cell nuclear antigen, Akt, mammalian target of rapamycin, and S6 Kinase in the transgenic mice as compared with the other 2 strains of PKD mice. Thus, overexpression of exogenous kidney-specific Ngal reduced cystic progression and prolonged the lifespan in PKD mice, was associated with reductions in interstitial fibrosis and proliferation, and augmented apoptosis.

Matrix-Stiffness-Regulated Inverse Expression of Krüppel-Like Factor 5 and Krüppel-Like Factor 4 in the Pathogenesis of Renal Fibrosis.

The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor (Klf)5 was the most up-regulated transcription factor accompanied by the down-regulation of Klf4 when cells were on stiff matrix. These changes were reversed by soft matrix via extracellular signal-regulated kinase (ERK) inactivation. Knockdown of Klf5 or forced expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive and negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/cyclin D1 axis was up-regulated and Klf4 was down-regulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated unilateral ureteral obstruction-induced tubular dilatation and proliferation, preserved Klf4, and suppressed the ERK/YAP1/Klf5/cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiologic functions and fibrosis progression.

Mechanosensitive store-operated calcium entry regulates the formation of cell polarity.

Ca(2+) -mediated formation of cell polarity is essential for directional migration which plays an important role in physiological and pathological processes in organisms. To examine the critical role of store-operated Ca(2+) entry, which is the major form of extracellular Ca(2+) influx in non-excitable cells, in the formation of cell polarity, we employed human bone osteosarcoma U2OS cells, which exhibit distinct morphological polarity during directional migration. Our analyses showed that Ca(2+) was concentrated at the rear end of cells and that extracellular Ca(2+) influx was important for cell polarization. Inhibition of store-operated Ca(2+) entry using specific inhibitors disrupted the formation of cell polarity in a dose-dependent manner. Moreover, the channelosomal components caveolin-1, TRPC1, and Orai1 were concentrated at the rear end of polarized cells. Knockdown of TRPC1 or a TRPC inhibitor, but not knockdown of Orai1, reduced cell polarization. Furthermore, disruption of lipid rafts or overexpression of caveolin-1 contributed to the downregulation of cell polarity. On the other hand, we also found that cell polarity, store-operated Ca(2+) entry activity, and cell stiffness were markedly decreased by low substrate rigidity, which may be caused by the disorganization of actin filaments and microtubules that occurs while regulating the activity of the mechanosensitive TRPC1 channel.

Mechanical coupling of cytoskeletal elasticity and force generation is crucial for understanding the migrating nature of keloid fibroblasts.

One of the key features of keloid is its fibroblasts migrating beyond the original wound border. During migration, cells not only undergo molecular changes but also mechanical modulation. This process is led by actin filaments serving as the backbone of intra-cellular force and transduces external mechanical signal via focal adhesion complex into the cell. Here, we focus on determining the mechanical changes of actin filaments and the spatial distribution of forces in response to changing chemical stimulations and during cell migration. Atomic force microscopy and micropost array detector are used to determine and compare the magnitude and distribution of filament elasticity and force generation in fibroblasts and keloid fibroblasts. We found both filament elasticity and force generation show spatial distribution in a polarized and migrating cell. Such spatial distribution is disrupted when mechano-signalling is perturbed by focal adhesion kinase inhibitor and in keloid fibroblasts. The demonstration of keloid pathology at the nanoscale highlights the coupling of cytoskeletal function with physical characters at the subcellular level and provides new research directions for migration-related disease such as keloid.

Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-ß1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-ß1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis.

SEPT12 mutations cause male infertility with defective sperm annulus.

Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis-specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation significantly reduced guanosine-5'-triphosphate (GTP) hydrolytic activity, and the p.Asp197Asn mutation (SEPT12(D197N)) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild-type SEPT12 in a dose-dependent manner. The patient carrying SEPT12(D197N) presented with oligoasthenozoospermia, whereas the SEPT12(T89M) patient had asthenoteratozoospermia. The characteristic sperm pathology of the SEPT12(D197N) patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss-of-function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation.

A tale of two collagen receptors, integrin ß1 and discoidin domain receptor 1, in epithelial cell differentiation.

As increase in collagen deposition is no longer taken as simply a consequence but, rather, an inducer of disease progression; therefore, the understanding of collagen signal transduction is fundamentally important. Cells contain at least two types of collagen receptors: integrins and discoidin domain receptors (DDRs). The integrin heterodimers α(1)ß(1), α(2)ß(1), α(10)ß(1), and α(11)ß(1) are recognized as the non-tyrosine kinase collagen receptors. DDR1 and 2, the tyrosine kinase receptors of collagen, are specifically expressed in epithelium and mesenchyme, respectively. While integrin ß(1) and DDR1 are both required for cell adhesion on collagen, their roles in epithelial cell differentiation during development and disease progression seem to counteract each other, with integrin ß(1) favoring epithelium mesenchyme transition (EMT) and DDR1 inducing epithelial cell differentiation. The in vitro evidence shows that the integrin ß(1) and DDR1 exert opposing actions in regulation of membrane stability of E-cadherin, which itself is a critical regulator of epithelial cell differentiation. Here, we review the functional roles of integrin ß(1) and DDR1 in regulation of epithelial cell differentiation during development and disease progression, and explore the underlining mechanisms regarding to the regulation of membrane stability of E-cadherin.

Ha-RasV12-Induced Multilayer Cellular Aggregates Is Mediated by Rac1 Activation Rather Than YAP Activation.

We demonstrate that Ha-RasV12 overexpression induces the nuclear translocation of Hippo effector Yes-associated protein (YAP) in MDCK cells via the hippo-independent pathway at the confluent stage. Ha-RasV12 overexpression leads to the downregulation of Caveolin-1 (Cav1) and the disruption of junction integrity. It has been shown that the disruption of actin belt integrity causes YAP nuclear translocation in epithelial cells at high density. Therefore, we hypothesized that Ha-RasV12-decreased Cav1 leads to the disruption of cell junction integrity, which subsequently facilitates YAP nuclear retention. We revealed that Ha-RasV12 downregulated Cav1 through the ERK pathway. Furthermore, the distribution and expression of Cav1 mediated the cell junction integrity and YAP nuclear localization. This suggests that the downregulation of Cav1 induced by Ha-RasV12 disrupted the cell junction integrity and promoted YAP nuclear translocation. We further indicated the consequence of Ha-RasV12-induced YAP activation. Surprisingly, the activation of YAP is not required for Ha-RasV12-induced multilayer cellular aggregates. Instead, Ha-RasV12 triggered the ERK-Rac pathway to promote cellular aggregate formation. Moreover, the overexpression of constitutively active Rac is sufficient to trigger cellular aggregation in MDCK cells at the confluent stage. This highlights that Rac activity is essential for cellular aggregates.

Mouse kidney progenitor cells accelerate renal regeneration and prolong survival after ischemic injury.

Acute tubular necrosis is followed by regeneration of damaged renal tubular epithelial cells, and renal stem cells are supposed to contribute to this process. The purpose of our study is to test the hypothesis that renal stem cells isolated from adult mouse kidney accelerate renal regeneration via participation in the repair process. A unique population of cells exhibiting characteristics consistent with renal stem cells, mouse kidney progenitor cells (MKPC), was isolated from Myh9 targeted mutant mice. Features of these cells include (1) spindle-shaped morphology, (2) self-renewal of more than 100 passages without evidence of senescence, and (3) expression of Oct-4, Pax-2, Wnt-4, WT-1, vimentin, alpha-smooth muscle actin, CD29, and S100A4 but no SSEA-1, c-kit, or other markers of more differentiated cells. MKPC exhibit plasticity as demonstrated by the ability to differentiate into endothelial cells and osteoblasts in vitro and endothelial cells and tubular epithelial cells in vivo. The origin of the isolated MKPC was from the interstitium of medulla and papilla. Importantly, intrarenal injection of MKPC in mice with ischemic injury rescued renal damage, as manifested by decreases in peak serum urea nitrogen, the infarct zone, and the necrotic injury. Seven days after the injury, some MKPC formed vessels with red blood cells inside and some incorporated into renal tubules. In addition, MKPC treatment reduces the mortality in mice after ischemic injury. Our results indicate that MKPC represent a multipotent adult stem cell population, which may contribute to the renal repair and prolong survival after ischemic injury.

Cigarette dependence questionnaire: development and psychometric testing with male smokers.

AIM: This paper is a report of a study conducted to develop and test a theoretically derived Cigarette Dependence Questionnaire for adult male smokers. BACKGROUND: Fagerstrom questionnaires have been used worldwide to assess cigarette dependence. However, these assessments lack any theoretical perspective. A theory-based approach is needed to ensure valid assessment. METHODS: In 2007, an initial pool of 103 Cigarette Dependence Questionnaire items was distributed to 109 adult smokers in Taiwan. Item analysis was conducted to select items for inclusion in the refined scale. The psychometric properties of the Cigarette Dependence Questionnaire were further evaluated 2007-08, when it was administered to 256 respondents and their saliva was collected and analysed for cotinine levels. Criterion validity was established through the Pearson correlation between the scale and saliva cotinine levels. Exploratory factor analysis was used to test construct validity. Reliability was determined with Cronbach's alpha coefficient and a 2-week test-retest coefficient. RESULTS: The selection of 30 items for seven perspectives was based on item analysis. One factor accounting for 44.9% of the variance emerged from the factor analysis. The factor was named as cigarette dependence. Cigarette Dependence Questionnaire scores were statistically significantly correlated with saliva cotinine levels (r = 0.21, P = 0.01). Cronbach's alpha was 0.95 and test-retest reliability using an intra-class correlation was 0.92. CONCLUSION: The Cigarette Dependence Questionnaire showed sound reliability and validity and could be used by nurses to set up smoking cessation interventions based on assessment of cigarette dependence.

Psychometric testing of the Chinese version of the Dimensions of Tobacco Dependence Scale.

AIMS: This paper reports on a study to establish the psychometric properties of the Chinese version of the dimensions of Tobacco Dependence Scale as these apply in Taiwan. BACKGROUND: The instruments for measuring cigarette dependence among adolescent smokers are not available in Taiwan. Therefore, it has not so far been possible to assess such dependence. DESIGN: Survey. METHOD: A sample comprising 293 adolescent smokers was recruited. They were drawn from nine senior high schools in middle and southern Taiwan. The Dimensions of Tobacco Dependence Scale and the Fagerstrom Test of nicotine dependence were administered, with saliva being subjected to salivary cotinine analysis using competitive enzyme immunoassay kits. RESULTS: The internal structure of the Dimensions of Tobacco Dependence Scale (DTDS) was first examined using exploratory factor analysis with promax rotation. By applying the parallel test as the means of selection, four criteria emerged from the factor analysis that accounted for 47.7% of the variance. Concerning criterion validity, the DTDS was found to have a positive and significant association with the Fagerstrom test of nicotine dependence scores (r = 0.58; p < 0.01) and with saliva cotinine levels (r = 0.30; p < 0.01). Cronbach's alpha values for the DTDS were 0.94. CONCLUSIONS: The study confirmed that the Chinese version of the DTDS is similar to the English version in that it is multidimensional and has consistent factor structures. And, as a result of the study, it was established that the Chinese version of the Dimensions of Tobacco Dependence Scale is a reliable and valid measure of tobacco dependence among adolescents in Taiwan. RELEVANCE TO CLINICAL PRACTICE: The DTDS can provide health professionals with a reference measure for assessing adolescents' tobacco dependence when applied in the context of a smoking cessation programme.

Dichotomy of the function of DDR1 in cells and disease progression.

Discoidin domain receptors DDR1 and DDR2 are collagen receptor tyrosine kinases that have many roles in tissue development and disease progression. Under physiological conditions, DDR1 is predominantly expressed in epithelial cells and functions to maintain cell differentiation and tissue homeostasis. A switch in expression from DDR1 to DDR2 occurs during epithelial-to-mesenchymal transition. However, opposite effects of DDR1 are reported to be involved in the progression of cancer and fibrotic diseases. Accumulating evidence suggests that DDR1 is involved in pro-metastasis and pro-survival signals. This review summarizes the roles of DDR1 in epithelial cell differentiation, cell migration, cancer progression and tissues fibrosis and highlights how the dichotomous functions of DDR1 may relevant to different cell types and statues. Elucidation of the underlying mechanism of the dichotomous functions of DDR1 will help to develop DDR1 as a therapeutic target.

Evaluating screening performances of the Fagerstrom tolerance questionnaire, the Fagerstrom test for nicotine dependence and the heavy smoking index among Taiwanese male smokers.

AIM: To compare screening performances of the Fagerstrom Tolerance Questionnaire (FTQ), the Fagerstrom test for nicotine dependence (FTND) and the Heavy Smoking Index (HSI) with a view to determining the optimum cutoff scores using biomarkers as standards. BACKGROUND: Previous studies proposed inconsistent cutoff scores for the FTQ, the FTND as signalling nicotine dependence and these scores were established by applying diverse standards. METHOD: Receiver operating characteristic (ROC) analyses were used in pursuit of the study's objectives. Two hundred and forty-five male smokers were recruited in 2005 from among those attending public health stations in southern Taiwan. The three self-report measures of nicotine dependence were compared with the saliva cotinine and expired carbon monoxide (CO). The expired CO level was tested by means of a Micro Smokerlyzer, while salivary cotinine was analysed using an enzyme-linked immunosorbent assay. RESULTS: The areas under the ROC curves for the FTQ, the FTND and the HSI were 0.71, 0.76 and 0.76 for the salivary cotinine and 0.71, 0.79 and 0.80 for the exhaled CO respectively. The sensitivity and specificity of the FTND and the HSI were slightly greater than those for the FTQ. The optimum cutoff scores for the FTQ, the FTND and the HSI as screening tools to establish nicotine dependence would be 5+, 4+ and 3+ respectively. CONCLUSION: The results indicate that the FTND and the HSI may be more efficacious than the FTQ in assessing nicotine dependence. Further research is needed to confirm these findings, especially among female smokers and for nicotine substitution trials. Relevance to clinical practice. To decrease tobacco-attributable morbidity and mortality, nurses and healthcare professionals need to implement effective smoking cessation interventions. The FTND and the HSI as well as their cutoff scores will be suitably used to assess nicotine dependence in these interventions.

Smoking characteristics and saliva cotinine levels in Taiwanese smokers: gender differences.

AIMS: The present study examined the smoking characteristics and a biomarker of a community-based sample of Taiwanese individuals, seeking to determine the correlates of nicotine dependence for each gender. BACKGROUND: Nicotine dependence is a key barrier to smoking cessation. Additionally, nicotine metabolism may influence smoking behaviour and dependence. Correlates of nicotine dependence and biochemical markers of smoking for the Taiwanese population have not yet been studied. METHODS: Participants were 402 men and 110 women smokers, who had recently smoked at least one cigarette every day over a recent year. All participants completed a questionnaire which included demographic data, smoking background, self-reported quantity of cigarettes per day and the Fagerstrom Test for Nicotine Dependence. The participants also provided saliva samples. RESULTS: Female smokers were younger, more likely to be unmarried and were on average higher educated than males. A higher percentage of females reported that family members smoked and tended to be younger at initiation and were also more likely to have attempted to quit on previous occasions. Males reported smoking significantly more cigarettes than females, however, there was no significant difference in the Fagerstrom Test for Nicotine Dependence scores between the two groups. The average saliva cotinine level was significantly higher for males than females (228.8 ng/ml vs. 94.6 ng/ml, p < 0.0001). Family and friends smoking were risk factors of nicotine dependence among both men and women smokers, but there was a stronger association of both factors on dependence levels for women. Being married is a risk factor for dependence for women but not for men. CONCLUSION: These findings support the evidence that smoking characteristics and a biochemical marker differ between the males and females. RELEVANCE TO CLINICAL PRACTICE: The results of the study are useful for health policymakers, assisting them in planning tobacco control activities in light of gender differences.

Mechanical forces in skin disorders.

Mechanical forces are known to regulate homeostasis of the skin and play a role in the pathogenesis of skin diseases. The epidermis consists of keratinocytes that are tightly adhered to each other by cell junctions. Defects in keratins or desmosomal/hemidesmosomal proteins lead to the attenuation of mechanical strength and formation of intraepidermal blisters in the case of epidermolysis bullosa simplex. The dermis is rich in extracellular matrix, especially collagen, and provides the majority of tensile force in the skin. Keloid and hypertrophic scar, which is the result of over-production of collagen by fibroblasts during the wound healing, are associated with extrinsic tensile forces and changes of intrinsic mechanical properties of the cell. Increasing evidences shows that stiffness of the skin environment determines the regenerative ability during wound healing process. Mechanotransduction pathways are also involved in the morphogenesis and cyclic growth of hair follicles. The development of androgenetic alopecia is correlated to tensile forces generated by the fibrous tissue underlying the scalp. Acral melanoma predominantly occurs in the weight-bearing area of the foot suggesting the role of mechanical stress. Increased dermal stiffness from fibrosis might be the cause of recessive dystrophic epidermolysis bullosa associated squamous cell carcinoma. Strategies to change the mechanical forces or modify the mechanotransduction signals may lead to a new way to treat skin diseases and promote skin regeneration.

Bcl-2 regulates store-operated Ca2+ entry to modulate ER stress-induced apoptosis.

Ca2+ plays a significant role in linking the induction of apoptosis. The key anti-apoptotic protein, Bcl-2, has been reported to regulate the movement of Ca2+ across the ER membrane, but the exact effect of Bcl-2 on Ca2+ levels remains controversial. Store-operated Ca2+ entry (SOCE), a major mode of Ca2+ uptake in non-excitable cells, is activated by depletion of Ca2+ in the ER. Depletion of Ca2+ in the ER causes translocation of the SOC channel activator, STIM1, to the plasma membrane. Thereafter, STIM1 binds to Orai1 or/and TRPC1 channels, forcing them to open and thereby allow Ca2+ entry. In addition, several anti-cancer drugs have been reported to induce apoptosis of cancer cells via the SOCE pathway. However, the detailed mechanism underlying the regulation of SOCE by Bcl-2 is not well understood. In this study, a three-amino acid mutation within the Bcl-2 BH1 domain was generated to verify the role of Bcl-2 in Ca2+ handling during ER stress. The subcellular localization of the Bcl-2 mutant (mt) is similar to that in the wild-type Bcl-2 (WT) in the ER and mitochondria. We found that mt enhanced thapsigargin and tunicamycin-induced apoptosis through ER stress-mediated apoptosis but not through the death receptor- and mitochondria-dependent apoptosis, while WT prevented thapsigargin- and tunicamycin-induced apoptosis. In addition, mt depleted Ca2+ in the ER lumen and also increased the expression of SOCE-related molecules. Therefore, a massive Ca2+ influx via SOCE contributed to caspase activation and apoptosis. Furthermore, inhibiting SOCE or chelating either extracellular or intracellular Ca2+ inhibited mt-mediated apoptosis. In brief, our results explored the critical role of Bcl-2 in Ca2+ homeostasis and the modulation of ER stress.

Caveolin-1 Controls Hyperresponsiveness to Mechanical Stimuli and Fibrogenesis-Associated RUNX2 Activation in Keloid Fibroblasts.

Keloids are pathological scars characterized by excessive extracellular matrix production that are prone to form in body sites with increased skin tension. CAV1, the principal coat protein of caveolae, has been associated with the regulation of cell mechanics, including cell softening and loss of stiffness sensing ability in NIH3T3 fibroblasts. Although CAV1 is present in low amounts in keloid fibroblasts (KFs), the causal association between CAV1 down-regulation and its aberrant responses to mechanical stimuli remain unclear. In this study, atomic force microscopy showed that KFs were softer than normal fibroblasts with a loss of stiffness sensing. The decrease of CAV1 contributed to the hyperactivation of fibrogenesis-associated RUNX2, a transcription factor germane to osteogenesis/chondrogenesis, and increased migratory ability in KFs. Treatment of KFs with trichostatin A, which increased the acetylation level of histone H3, increased CAV1 and decreased RUNX2 and fibronectin. Trichostatin A treatment also resulted in cell stiffening and decreased migratory ability in KFs. Collectively, these results suggest a role for CAV1 down-regulation in linking the aberrant responsiveness to mechanical stimulation and extracellular matrix accumulation with the progression of keloids, findings that may lead to new developments in the prevention and treatment of keloid scarring.

Mechanotransduction of matrix stiffness in regulation of focal adhesion size and number: reciprocal regulation of caveolin-1 and ß1 integrin.

Focal adhesion (FA) assembly, mediated by integrin activation, responds to matrix stiffness; however, the underlying mechanisms are unclear. Here, we showed that ß1 integrin and caveolin-1 (Cav1) levels were decreased with declining matrix stiffness. Soft matrix selectively downregulated ß1 integrin by endocytosis and subsequent lysosomal degradation. Disruption of lipid rafts with methyl-ß-cyclodextrin or nystatin, or knockdown of Cav1 by siRNA decreased cell spreading, FA assembly, and ß1 integrin protein levels in cells cultured on stiff matrix. Overexpression of Cav1, particularly the phospho-mimetic mutant Cav1-Y14D, averted soft matrix-induced decreases in ß1 integrin protein levels, cell spreading, and FA assembly in NMuMG cells. Interestingly, overexpression of an auto-clustering ß1 integrin hindered soft matrix-induced reduction of Cav1 and cell spreading, which suggests a reciprocal regulation between ß1 integrin and Cav1. Finally, co-expression of this auto-clustering ß1 integrin and Cav1-Y14D synergistically enhanced cell spreading, and FA assembly in HEK293T cells cultured on either stiff ( > G Pa) or soft (0.2 kPa) matrices. Collectively, these results suggest that matrix stiffness governs the expression of ß1 integrin and Cav1, which reciprocally control each other, and subsequently determine FA assembly and turnover.