scholarly journals Conditional Ablation of β1 Integrin in Skin

2000 ◽  
Vol 150 (5) ◽  
pp. 1149-1160 ◽  
Author(s):  
Srikala Raghavan ◽  
Christoph Bauer ◽  
Gina Mundschau ◽  
Qingqin Li ◽  
Elaine Fuchs
Keyword(s):  
Hair Follicle ◽  
Terminal Differentiation ◽  
Conditional Knockout ◽  
Matrix Assembly ◽  
Culture Studies ◽  
Laminin 5 ◽  
Stem Cell Maintenance ◽  
Epidermal Proliferation ◽  
Knockout Technology

The major epidermal integrins are α3β1 and hemidesmosome-specific α6β4; both share laminin 5 as ligand. Keratinocyte culture studies implicate both integrins in adhesion, proliferation, and stem cell maintenance and suggest unique roles for αβ1 integrins in migration and terminal differentiation. In mice, however, whereas ablation of α6 or β4 results in loss of hemidesmosomes, epidermal polarity, and basement membrane (BM) attachment, ablation of α3 only generates microblistering due to localized internal shearing of BM. Using conditional knockout technology to ablate β1 in skin epithelium, we have uncovered biological roles for αβ1 integrins not predicted from either the α3 knockout or from in vitro studies. In contrast to α3 null mice, β1 mutant mice exhibit severe skin blistering and hair defects, accompanied by massive failure of BM assembly/organization, hemidesmosome instability, and a failure of hair follicle keratinocytes to remodel BM and invaginate into the dermis. Although epidermal proliferation is impaired, a spatial and temporal program of terminal differentiation is executed. These results indicate that β1's minor partners in skin are important, and together, αβ1 integrins are required not only for extracellular matrix assembly but also for BM formation. This, in turn, is required for hemidesmosome stability, epidermal proliferation, and hair follicle morphogenesis. However, β1 downregulation does not provide the trigger to terminally differentiate.

In Vitro Proliferation and Differentiation of Human Hair Follicle Stem Cells on Chitosan-Skin Regenerating Template

2015 ◽  
Vol 05 (999) ◽  
pp. 1-1
Author(s):  
Abu Bakar Mohd Hilmi ◽  
Mohd Noor Norhayati ◽  
Ahmad Sukari Halim ◽  
Chin Keong Lim ◽  
Zulkifli Mustafa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hair Follicle ◽  
Human Hair ◽  
Human Hair Follicle ◽  
In Vitro Proliferation ◽  
Proliferation And Differentiation ◽  
Hair Follicle Stem Cells ◽  
Follicle Stem Cells

scholarly journals Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Anthony R. Anzell ◽  
Garrett M. Fogo ◽  
Zoya Gurm ◽  
Sarita Raghunayakula ◽  
Joseph M. Wider ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Ischemia Reperfusion Injury ◽  
Mitochondrial Dynamics ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fission ◽  
Conditional Knockout ◽  
Mitochondrial Morphology ◽  
Primary Neurons ◽  
Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

In vitro assessment of food-derived-glucose bioaccessibility and bioavailability in bicameral cell culture system

2020 ◽  
Vol 45 (5) ◽  
pp. 631-637
Author(s):  
Cansu Ozel-Tasci ◽  
Gozde Pilatin ◽  
Ozgur Edeer ◽  
Sukru Gulec
Keyword(s):  
Cell Culture ◽  
Culture System ◽  
Metabolic Diseases ◽  
Enzymatic Digestion ◽  
Cell Culture System ◽  
Culture Studies ◽  
Experimental Approaches ◽  
In Vitro Assessment

AbstractBackgroundFunctional foods can help prevent metabolic diseases, and it is essential to evaluate functional characteristics of foods through in vitro and in vivo experimental approaches.ObjectiveWe aimed to use the bicameral cell culture system combined with the in vitro digestion to evaluate glucose bioavailability.Materials and methodsCake, almond paste, and pudding were modified by adding fiber and replacing sugar with sweeteners and polyols. Digestion process was modeled in test tubes. Rat enterocyte cells (IEC-6) were grown in a bicameral cell culture system to mimic the physiological characteristics of the human intestine. The glucose bioaccessibility and cellular glucose efflux were measured by glucose oxidase assay.Results and discussionThe glucose bioaccessibilities of modified foods were significantly lower (cake: 2.6 fold, almond paste: 9.2 fold, pudding 2.8 fold) than the controls. Cellular glucose effluxes also decreased in the modified cake, almond paste, and pudding by 2.2, 4, and 2 fold respectively compared to their controls.ConclusionOur results suggest that combining in vitro enzymatic digestion with cell culture studies can be a practical way to test in vitro glucose bioaccessibility and bioavailability in functional food development.

scholarly journals Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia

Blood ◽  
2020 ◽  
Vol 136 (2) ◽  
pp. 210-223 ◽  
Author(s):  
Eun Ji Gang ◽  
Hye Na Kim ◽  
Yao-Te Hsieh ◽  
Yongsheng Ruan ◽  
Heather A. Ogana ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Underlying Mechanism ◽  
Conditional Knockout

Abstract Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

scholarly journals Isolation of erythropoietin-sensitive cells from Friend virus-infected marrow cultures: characteristics of the erythropoietin response

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 751-758 ◽  
Author(s):  
M Bondurant ◽  
M Koury ◽  
SB Krantz ◽  
T Blevins ◽  
DT Duncan
Keyword(s):  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Precursor Cells ◽  
Erythroid Cells ◽  
Friend Virus ◽  
Isolated Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Erythroid Precursor ◽  
Short Time

Abstract Murine erythroid precursor cells, stimulated to proliferate in vitro in the absence of added erythropoietin (EP) by the anemia strain of Friend virus (FVA), will subsequently respond to EP by complete erythrocyte differentiation. If not exposed to EP, the erythroid cells divide for about 120 hr in culture, and they maintain the potential for full differentiation in response to EP added at any time during the period from 72 to 120 hr. Between 96 and 120 hr of culture without added EP, the EP-sensitive erythroid precursor cells that have formed discrete erythroid bursts can be isolated in relatively large numbers from such cultures by plucking with a Pasteur pipette. The addition of EP initiates the final stages of erythroid differentiation, including heme synthesis in 70%-80% of these isolated cells. With respect to homogeneity of the precursor cells, quantity of EP-responsive cells obtainable, and uniformity of EP responsiveness, this system is uniquely favorable for biochemical studies of the late differentiation effects of EP. The overall changes in gene expression accompanying EP- induced terminal differentiation were examined by two-dimensional gel electrophoresis of proteins labeled for a short time with radioactive amino acids. Several new proteins are synthesized in these erythroid cells during terminal differentiation, but the number is a very small percentage of the total number of proteins being made. Thus, in this system, the effect of EP is to initiate expression of a small group of genes, including those for globins, spectrin, and other proteins involved in the final stages of erythroid differentiation.

scholarly journals A Mouse Model for Studying Stem Cell Effects on Regeneration of Hair Follicle Outer Root Sheaths

2020 ◽  
Vol 15 (1) ◽  
pp. 41-50
Author(s):  
Jingxu Guo ◽  
Shuwei Li ◽  
Hongyang Wang ◽  
Tinghui Wu ◽  
Zhenhui Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mouse Model ◽  
Hair Follicle ◽  
Smooth Muscle Actin ◽  
Hair Follicles ◽  
Alpha Smooth Muscle Actin ◽  
Papillary Structure ◽  
Glass Membrane

AbstractObjectiveStem cells hold promise for treating hair loss. Here an in vitro mouse model was developed using outer root sheaths (ORSs) isolated from hair follicles for studying stem cell-mediated dermal papillary regeneration.MethodsUnder sterile conditions, structurally intact ORSs were isolated from hair follicles of 3-day-old Kunming mice and incubated in growth medium. Samples were collected daily for 5 days. Stem cell distribution, proliferation, differentiation, and migration were monitored during regeneration.ResultsCell proliferation began at the glass membrane periphery then spread gradually toward the membrane center, with the presence of CD34 and CD200 positive stem cells involved in repair initiation. Next, CD34 positive stem cells migrated down the glass membrane, where some participated in ORS formation, while other CD34 cells and CD200 positive cells migrated to hair follicle centers. Within the hair follicle matrix, stem cells divided, grew, differentiated and caused outward expansion of the glass membrane to form a dermal papillary structure containing alpha-smooth muscle actin. Neutrophils attracted to the wound site phagocytosed bacterial and cell debris to protect regenerating tissue from infection.ConclusionIsolated hair follicle ORSs can regenerate new dermal papillary structures in vitro. Stem cells and neutrophils play important roles in the regeneration process.

scholarly journals Sirt1 deacetylates and stabilizes p62 to promote hepato-carcinogenesis

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Lifeng Feng ◽  
Miaoqin Chen ◽  
Yiling Li ◽  
Muchun Li ◽  
Shiman Hu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Overall Survival ◽  
Cell Growth ◽  
Knockout Mice ◽  
Liver Tumors ◽  
Underlying Mechanism ◽  
Carcinoma Cells ◽  
Conditional Knockout

Abstractp62/SQSTM1 is frequently up-regulated in many cancers including hepatocellular carcinoma. Highly expressed p62 promotes hepato-carcinogenesis by activating many signaling pathways including Nrf2, mTORC1, and NFκB signaling. However, the underlying mechanism for p62 up-regulation in hepatocellular carcinoma remains largely unclear. Herein, we confirmed that p62 was up-regulated in hepatocellular carcinoma and its higher expression was associated with shorter overall survival in patients. The knockdown of p62 in hepatocellular carcinoma cells decreased cell growth in vitro and in vivo. Intriguingly, p62 protein stability could be reduced by its acetylation at lysine 295, which was regulated by deacetylase Sirt1 and acetyltransferase GCN5. Acetylated p62 increased its association with the E3 ligase Keap1, which facilitated its poly-ubiquitination-dependent proteasomal degradation. Moreover, Sirt1 was up-regulated to deacetylate and stabilize p62 in hepatocellular carcinoma. Additionally, Hepatocyte Sirt1 conditional knockout mice developed much fewer liver tumors after Diethynitrosamine treatment, which could be reversed by the re-introduction of exogenous p62. Taken together, Sirt1 deacetylates p62 at lysine 295 to disturb Keap1-mediated p62 poly-ubiquitination, thus up-regulating p62 expression to promote hepato-carcinogenesis. Therefore, targeting Sirt1 or p62 is a reasonable strategy for the treatment of hepatocellular carcinoma.

Gli2-regulated activation of hepatic stellate cells and liver fibrosis by TGF-β signaling

2021 ◽  
Author(s):  
Junyan Yan ◽  
Baowei Hu ◽  
Wenjie Shi ◽  
Xiaoyi Wang ◽  
Jiayuan Shen ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Conditional Knockout ◽  
Rna Seq ◽  
Expression Levels ◽  
Liver Fibrogenesis ◽  
Hh Signaling ◽  
Signaling Activity

The Hedgehog (Hh) signaling pathway is correlated with hepatic stellate cells (HSCs) activation and liver fibrosis. Gli2 is a key transcription effector of Hh signaling. However, the role of Gli2 in HSC-mediated liver fibrosis progression is largely unknown. In the present study, we investigated the effect of Gli2 on liver fibrogenesis and its possible mechanism using conditional knockout (cKO) Gli2 mice and HSC models. Wild-type (WT) and GFAP-CreERT;Gli2flox/flox male mice were exposed to CCl4 for one month to induce liver fibrosis. Primary HSCs were isolated from mice and the transition of HSCs into a myofibroblastic phenotype was evaluated. Livers from mice underwent histological, immunohistochemical, and immunofluorescence analyses. The expression levels of proteins and genes were evaluated by Western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RNA-seq was used to screen differentially expressed genes. Results showed that CCl4 treatment induced liver fibrosis, promoted HSCs activation and proliferation, and up-regulated Hh signaling activity. The cKO of Gli2 in GFAP-CreERT;Gli2flox/flox mice decreased liver fibrosis as well as HSC activation and proliferation. In vitro studies showed that KO of Gli2 in HSCs blocked cell proliferation and activation by decrease of cyclin D1/D2 expression. The RNA-seq results revealed that the expression levels TGF-β1 ligands were down-regulated in Gli2 KO HSCs. Furthermore, overexpression of Gli2 rescued proliferation and activation of HSCs by up-regulation of TGF-β signaling activity. Our data demonstrated that Gli2 regulated HSC activation and liver fibrosis by TGF-β signaling, thus providing support for future Gli2-based investigations of liver fibrosis therapy.

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