scholarly journals Novel human ZAKI-4 isoforms: hormonal and tissue-specific regulation and function as calcineurin inhibitors

2002 ◽  
Vol 367 (2) ◽  
pp. 459-466 ◽  
Author(s):  
Xia CAO ◽  
Fukushi KAMBE ◽  
Takashi MIYAZAKI ◽  
Devanand SARKAR ◽  
Sachiko OHMORI ◽  
...  
Keyword(s):  
Human Skin ◽  
Single Gene ◽  
Human Fibroblasts ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Tissue Specific ◽  
Conserved Motif ◽  
N Terminus ◽  
Specific Regulation ◽  
Calcineurin Activity

We identified a thyroid hormone [3,5,3′-tri-iodothyronine (T3)]-responsive gene, ZAKI-4, in cultured human skin fibroblasts. It belongs to a family of genes that encode proteins containing a conserved motif. The motif binds to calcineurin and inhibits its phosphatase activity. In the present study, we have demonstrated three different ZAKI-4 transcripts, α, β1 and β2, in human brain by 5′- and 3′-RACE (rapid amplification of cDNA ends). The α transcript was identical with the one that we originally cloned from human fibroblasts and the other two are novel. The three transcripts are generated by alternative initiation and splicing from a single gene on the short arm of chromosome 6. It is predicted that β1 and β2 encode an identical protein product, β, which differs from α in its N-terminus. Since α and β contain an identical C-terminal region harbouring the conserved motif, both isoforms are suggested to inhibit calcineurin activity. Indeed, each isoform associates with calcineurin A and inhibits its activity in a similar manner, suggesting that the difference in N-terminus of each isoform does not affect the inhibitory function on calcineurin. An examination of the expression profile of the three transcripts in 12 human tissues revealed that the α transcript is expressed exclusively in the brain, whereas β transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both α and β transcripts, raising the question of which transcript is up-regulated by T3. It was revealed that T3 markedly induced the expression of α isoform but not of β. This T3-mediated increase in the α isoform was associated with a significant decrease in endogenous calcineurin activity. These results suggest that the expression of ZAKI-4 isoforms is subjected to distinct hormonal as well as tissue-specific regulation, constituting a complex signalling network through inhibition of calcineurin.

The Effects of Baicalin Against UVA-Induced Photoaging in Skin Fibroblasts

2014 ◽  
Vol 42 (03) ◽  
pp. 709-727 ◽  
Author(s):  
Wei Min ◽  
Xin Liu ◽  
Qihong Qian ◽  
Bingjiang Lin ◽  
Di Wu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Telomere Length ◽  
Human Skin ◽  
Human Fibroblasts ◽  
Underlying Mechanism ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Uva Irradiation ◽  
Anti Oxidant ◽  
Uva Radiation

Ultraviolet A (UVA) radiation contributes to skin photoaging. Baicalin, a plant-derived flavonoid, effectively absorbs UV rays and has been shown to have anti-oxidant and anti-inflammatory properties that may delay the photoaging process. In the current study, cultured human skin fibroblasts were incubated with 50 μg/ml baicalin 24 hours prior to 10 J/cm2UVA irradiation. In order to examine the efficacy of baicalin treatment in delaying UVA-induced photoaging, we investigated aging-related markers, cell cycle changes, anti-oxidant activity, telomere length, and DNA damage markers. UVA radiation caused an increased proportion of β-Gal positive cells and reduced telomere length in human skin fibroblasts. In addition, UVA radiation inhibited TGF-β1 secretion, induced G1 phase arrest, reduced SOD and GSH-Px levels, increased MDA levels, enhanced the expression of MMP-1, TIMP-1, p66, p53, and p16 mRNA, reduced c-myc mRNA expression, elevated p53 and p16 protein expression, and reduced c-myc protein expression. Baicalin treatment effectively protected human fibroblasts from these UVA radiation-induced aging responses, suggesting that the underlying mechanism involves the inhibition of oxidative damage and regulation of the expression of senescence-related genes, including those encoding for p53, p66Shcand p16.

scholarly journals Protein Signature of Human Skin Fibroblasts Allows the Study of the Molecular Etiology of Rare Neurological Diseases

2020 ◽  
Author(s):  
Andreas Hentschel ◽  
Artur Czech ◽  
Ute Münchberg ◽  
Erik Freier ◽  
Ulrike Schara-Schmidt ◽  
...  
Keyword(s):  
Human Skin ◽  
Muscle Biopsy ◽  
Neurological Diseases ◽  
Human Fibroblasts ◽  
Skin Fibroblasts ◽  
Proteomic Profiling ◽  
Human Skin Fibroblasts ◽  
Disease Etiology ◽  
Molecular Etiology

Abstract Background: The elucidation of pathomechanisms leading to the manifestation of rare (genetically caused) neurological diseases including neuromuscular diseases (NMD) represents an important step toward the understanding of the genesis of the respective disease and might help to define starting points for (new) therapeutic intervention concepts. However, these “discovery studies” are often limited by the availability of human biomaterial. Moreover, given that results of next-generation-sequencing approaches frequently result in the identification of ambiguous variants, testing of their pathogenicity is crucial but also depending on patient-derived material. Results: To systematically address the question if human skin fibroblasts might serve as valuable biomaterial for (molecular) studies of NMD, using proteomic profiling, we generated a protein library by decreasing protein complexity via pH8-based sample fractionation: cataloguing of 8280 proteins revealed the expression of a variety of such linked to genetic forms of motoneuron diseases, congenital myasthenic syndromes, neuropathies and muscle disorders. In silico-based pathway analyses revealed expression of a variety of proteins involved in muscle contraction and such decisive for neuronal function and maintenance suggesting the suitability of human skin fibroblasts to study the etiology of NMD. Based on these findings, next we aimed to further demonstrate the suitability of this in vitro model to study NMD by a use case: utilizing a data independent acquisition approach, the proteomic signature of whole protein extracts of fibroblasts derived from an Allgrove-patient was studied. Paradigmatic dysregulated proteins were confirmed in muscle biopsy of the patient and protein-functions could be linked to neurological symptoms known for this disease. Moreover, protoemic investigation of nuclear protein composition allowed the identification of protein-dysregulations according with structural perturbations observed in the muscle biopsy. As proteomic data suggested a perturbed lipid homeostasis, BODIPY-staining was performed on fibroblasts and coherent anti-stokes Raman scattering microscopy on muscle biopsy. Results of both investigations suggest altered lipid storage as part of the underlying disease-etiology. Conclusions: our combined data reveal that human fibroblasts may serve as an in vitro system to study the molecular etiology of rare neurological diseases exemplified on Allgrove syndrome in an unbiased fashion.

Insulin and Serum-stimulated Hexose Transport in In Vitro Aged Cultured Human Skin Fibroblasts

1986 ◽  
Vol 5 (3) ◽  
pp. 157-164 ◽  
Author(s):  
Ralph J. Germinario ◽  
Maureen Oliveira ◽  
Susannia Manuel
Keyword(s):  
Human Skin ◽  
Human Fibroblasts ◽  
Skin Fibroblasts ◽  
Proliferative Potential ◽  
Hexose Transport ◽  
Human Skin Fibroblasts ◽  
Short Term ◽  
Cultured Human Fibroblasts ◽  
Insulin Responses

ABSTRACTInsulin (0.67 μM) and serum (10% v/v) stimulated hexose transport did not change with in vitro ageing of cultured human skin fibroblasts. Additionally, dexamethasone amplification of the insulin-stimulated response which yielded responses to near physiological concentrations of insulin (i.e. 6.7 nM), showed no changes with in vitro ageing. Basal (i.e. nonstimulated) hexose transport, either in the presence or absence of dexamethasone, did not change with in vitro ageing. The in vitro loss of proliferative potential during in vitro ageing of cultured human fibroblasts probably does not involve changes in short term insulin responses (e.g. stimulation of hexose transport).

Histamine receptors in human fibroblasts: inositol phosphates, Ca2+, and cell growth

1990 ◽  
Vol 258 (3) ◽  
pp. C533-C543 ◽  
Author(s):  
C. L. Johnson ◽  
C. G. Johnson ◽  
E. Bazan ◽  
D. Garver ◽  
E. Gruenstein ◽  
...  
Keyword(s):  
Human Skin ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Human Fibroblasts ◽  
Physiological Role ◽  
Skin Fibroblasts ◽  
Phosphoinositide Metabolism ◽  
Human Skin Fibroblasts ◽  
H1 Receptor

Histamine stimulated inositol phosphate formation by human skin fibroblasts. The effect of histamine was reduced but still readily apparent in the absence of extracellular Ca2+. Histamine caused a transient increase in intracellular free Ca2+ as detected by indo-1 and fura-2 fluorescence studies on cell populations and on individual cells. Similar increases were observed in the absence of extracellular Ca2+, indicating that the effect was primarily due to mobilization of Ca2+ from intracellular stores, presumably by inositol trisphosphate (IP3). The effects of histamine on phosphoinositide metabolism and intracellular Ca2+ were inhibited by pretreatment of the cells with phorbol esters, suggesting that the histamine receptor in fibroblasts is subject to feedback regulation by protein kinase C. Histamine inhibited the incorporation of [3H]-thymidine into DNA. The effects of histamine on inositol phosphate formation, intracellular Ca2+, and thymidine incorporation were blocked by the H1 receptor antagonist mepyramine. Our results indicate that human skin fibroblasts have H1 receptors coupled to the formation of inositol phosphates and mobilization of intracellular Ca2+. We suggest that this H1 receptor also mediates a block of the cell cycle and that histamine may play a physiological role in the regulation of fibroblast proliferation.

scholarly journals Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation.

1991 ◽  
Vol 114 (2) ◽  
pp. 337-342 ◽  
Author(s):  
R Panet ◽  
H Atlan
Keyword(s):  
Cell Proliferation ◽  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Human Skin ◽  
Human Fibroblasts ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Mitogenic Signal

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts.

scholarly journals Studies in porphyria. IV. Expression of the gene defect of acute intermittent porphyria in cultured human skin fibroblasts and amniotic cells: prenatal diagnosis of the porphyric trait.

1975 ◽  
Vol 142 (3) ◽  
pp. 722-731 ◽  
Author(s):  
S Sassa ◽  
G Solish ◽  
R D Levere ◽  
A Kappas
Keyword(s):  
Prenatal Diagnosis ◽  
Human Skin ◽  
Human Fibroblasts ◽  
Acute Intermittent Porphyria ◽  
Skin Fibroblasts ◽  
Gene Defect ◽  
Human Skin Fibroblasts ◽  
Cell Extracts ◽  
S Deficiency ◽  
Deficient Level

The gene lesion of the porphyrin-heme synthetic pathway in acute intermittent porphyria (AIP) is reflected in a deficient level of activity of the cytosol enzyme uroporphyrinogen I synthetase (URO-S). A marked URO-S deficiency has been demonstrated in the liver and in circulating erythrocytes of individuals with both active and latent AIP. This enzymic abnormality accounts for the excessive production and excretion into urine of the porphyrin precursors, lamda-aminolevulinic acid (ALA) and porphobilinogen (PBG) in AIP subjects. In this study, utilizing cell culture techniques, a marked URO-S deficiency has also been demonstrated in skin fibroblasts from AIP patients and in cells derived through aminocentesis from an approximately 17-wk old fetus. The prenatal diagnosis of the AIP trait in this fetus was confirmed postnatally by the demonstration in the child of a deficient level of erythrocyte URO-S activity which was comparable to those found in her AIP mother and affected sibling and which was approximately one-half the levels characterizing her normal father and aunt and a second unaffected sibling. The identification of the URO-S deficiency in cultured human fibroblasts from AIP patients was facilitated by a newly developed, sensitive assay for the enzyme activity. In this assay, the ability of such cells to convert ALA to protoporphyrin was quantitated; in the sequence of reactions involved in this transformation, URO-S is limiting so that the gene defect of AIP could be simply and precisely determined by appropriate spectrofluorometry of cell extracts. The technique described has distinct advantages over the direct enzymatic assay for URO-S activity in cultured human skin fibroblasts and permits clear differentiation of AIP carrier from normal individuals.

117-LB: Nonviral Direct Reprogramming of Human Skin Fibroblasts into Hormone-Expressing ß-Like Cell Clusters

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 117-LB
Author(s):  
LUKE R. LEMMERMAN ◽  
MARIA ANGELICA RINCON-BENAVIDES ◽  
SARAH A. TERSEY ◽  
BRITANI N. BLACKSTONE ◽  
HEATHER M. POWELL ◽  
...  
Keyword(s):  
Human Skin ◽  
Skin Fibroblasts ◽  
Direct Reprogramming ◽  
Human Skin Fibroblasts ◽  
Cell Clusters

Protective Effects of Green Tea Seed Extract against UVB-irradiated Human Skin Fibroblasts

2014 ◽  
Vol 43 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Ok Kyung Kim ◽  
Da-Eun Nam ◽  
Min-Jae Lee ◽  
Namgil Kang ◽  
Jae-Youn Lim ◽  
...  
Keyword(s):  
Human Skin ◽  
Green Tea ◽  
Seed Extract ◽  
Skin Fibroblasts ◽  
Protective Effects ◽  
Human Skin Fibroblasts
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