Expression Of Differentiation-related Proteins Research Articles

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds.

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.

Danshensu methyl ester enhances autophagy to attenuate pulmonary fibrosis by targeting lncIAPF-HuR complex.

Pulmonary fibrosis is an irreversible fibrotic process that has a high mortality rate and limited treatment options; thus, developing a novel therapeutic drug is critical. In this study, we synthesized danshensu methyl ester (DME) and explored its anti-pulmonary fibrotic ability on TGF-β1-stimulated lung fibroblast in vitro and on bleomycin-induced pulmonary fibrosis in vivo. Results showed that DME decreased the expression of differentiation-related proteins, including fibroblast activation protein 1 (FAP1) and S100 calcium-binding protein A4 (S100A4), and fibrotic markers, such as a-SMA, vimentin, and collagen in vivo and in vitro. In addition, DME markedly repressed myofibroblast proliferation and migration. Mechanistically, chromatin immunoprecipitation-PCR, RNA immunoprecipitation, half-life, and other experiments revealed that DME inhibited activating transcription factor 3 expression via TGF-β1 signal transduction leading to a decrease in lncIAPF transcription and stability. Moreover, DME blocked human antigen R (HuR) nucleocytoplasmic translocation and promoted its degradation via downregulating lncIAPF, which markedly decreased the expression of HuR target genes such as negative autophagic regulators (EZH2, STAT1, and FOXK1). Collectively, our results demonstrated that DME enhanced autophagy to attenuate pulmonary fibrosis via downregulating the lncIAPF-HuR-mediated autophagic axis and the lncIAPF-HuR complex can be the target for drug action.

Knock-down of filaggrin influences the mitogen-activated protein kinases signaling pathway in normal human epidermal keratinocytes.

Filaggrin is an essential structural protein of the stratum corneum binding to the keratin intermediate filaments to form a dense protein-lipid matrix. However, the function of filaggrin in epidermal terminal differentiation is not completely understood. To evaluate the effects of filaggrin on normal human epidermal keratinocytes (NHEKs) and to investigate the relevant mechanisms. Short hairpin RNA (shRNA) technology was used to knock-down filaggrin in normal human epidermal keratinocytes (NHEKs). Western blot and real-time quantitative PCR (qRT-PCR) were performed to detect expression of filaggrin, differentiation-related proteins and MAPK-related proteins. Filaggrin was successfully knocked down in NHEKs (99% efficiency). We found that the lack of filaggrin significantly decreased the expression of some differentiation-related proteins, including Cytokeratin 5 protein, Cytokeratin 14 protein, ST14 protein and SPRR3 protein (P<0.05). In addition, filaggrin knock-down significantly decreased expression of p-p38, p-ERK1/2, p-JNK, p-Akt, and p-NF-κB in NHEKs. Our study shows that filaggrin regulates epidermal terminal differentiation and impairs MAPK signaling pathway in normal human epidermal keratinocytes.

MAPK Pathway Involved in Epidermal Terminal Differentiation of Normal Human Epidermal Keratinocytes.

ObjectiveTo investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway in epidermal terminal differentiation.MethodsThe MAPK pathways (p38, ERK1/2, JNK) were inhibited by SB203580, PD98059, and SP600125 in normal human epidermal keratinocytes (NHEKs), respectively. Western blotting assays were performed to detect expression of filaggrin and differentiation-related proteins. The mRNA expressions of differentiation-related proteins were detected by real-time quantitative PCR (qRT-PCR).ResultsInhibition of MAPK pathway by SB203580, PD98059, and SP600125 resulted in significant reduction of filaggrin expression in NHEKs. Inhibition of the p38 MAPK pathway decreased the expression of differentiation-related proteins (cytokeratin 5, cytokeratin 14, ST14, and SPRR3), Akt, and NF-κB. Inhibition of JNK also suppressed expression of cytokeratin 14, SPRR3, Akt, and NF-κB. However, inhibition of ERK1/2 merely decreased expression of SPRR3 and Akt.ConclusionMAPK pathways regulates epidermal terminal differentiation in NHEKs. The p38 signaling pathway plays an especially important role.

Oroxylin A, a natural anticancer flavonoid compound, induces differentiation of t(8;21)-positive Kasumi-1 and primary acute myeloid leukemia cells.

AML1/ETO fusion gene is one of disease-causing genes of t(8;21)-positive acute myeloid leukemia (AML). Oroxylin A (OA) has showed anticancer effects on other cancer cells. Here, studies were conducted to determine the antileukemia effect of OA on t(8;21)-positive AML cells in vitro and in vivo. The effects of OA on cell viability of t(8;21)-positive Kasumi-1 and primary AML cells were analyzed by MTT assay. Cell differentiation was examined by NBT reduction assay, flow cytometry analysis for CD11b/CD14, and Giemsa stain. Protein expressions were determined by Western blots. Immunofluorescence assay was used to verify the effect of OA on HDAC-1 expression in vivo. Immunohistochemical staining was applied to evaluate leukemic infiltration of AML-bearing NOD/SCID mice. OA enhanced NBT reduction activity and CD11b/CD14 expression of AML1/ETO-positive AML cells markedly. Results of Giemsa staining also demonstrated that OA could induce the morphologic changes with reduction of nuclear/cytoplasmic ratios, suggesting the cell differentiation induced by OA. Further study showed that OA decreased the expression of fusion protein AML1/ETO and down-regulated HDAC-1 protein levels in vitro and in vivo. Moreover, OA increased the expression of differentiation-related proteins C/EBPα and P21. Acetylation levels of histones were also advanced obviously after treatment of OA. In vivo study indicated that OA could prolong the survival of AML-bearing NOD/SCID mice and reduce leukocytic infiltration of the spleen. All these results suggested that OA might be a novel candidate agent for differentiation therapy for AML1/ETO-positive AML and the mechanism required further investigation.

Mitogen-activated protein kinase activator with WD40 repeats (MAWD) and MAWD-binding protein induce cell differentiation in gastric cancer.

BackgroundOur previous proteomic analysis revealed that mitogen-activated protein kinase activator with WD40 repeats (MAWD) and MAWD-binding protein (MAWBP) were downregulated in gastric cancer (GC) tissues. These proteins interacted and formed complexes in GC cells. To investigate the role of MAWD and MAWBP in GC differentiation, we analyzed the relationship between MAWD/MAWBP and clinicopathologic characteristics of GC tissues and examined the expression of E-cadherin and pepsinogen C (PGC)—used as gastric mucosa differentiation markers—in MAWD/MAWBP-overexpressing GC cells and xenografts.MethodsWe measured MAWD, MAWBP, transforming growth factor-beta (TGF-beta), E-cadherin, and PGC expression in 223 GC tissues and matched-adjacent normal tissues using tissue microarray and immunohistochemistry (IHC) analyses, and correlated these expression levels with clinicopathologic features. MAWD and MAWBP were overexpressed alone or together in SGC7901 cells and then E-cadherin, N-cadherin, PGC, Snail, and p-Smad2 levels were determined using western blotting, semiquantitative RT-PCR, and immunofluorescence analysis. Alkaline phosphatase (AKP) activity was measured to investigate the differentiation level of various transfected cells, and the transfected cells were used in tumorigenicity assays and for IHC analysis of protein expression in xenografts.ResultsMAWD/MAWBP positive staining was significantly lower in GC tissues than in normal samples (P < 0.001), and the expression of these proteins was closely correlated with GC differentiation grade. Kaplan–Meier survival curves indicated that low MAWD and MAWBP expression was associated with poor patient survival (P < 0.05). The differentiation-related proteins E-cadherin and PGC were expressed in GC tissues at a lower level than in normal tissues (P < 0.001), but were upregulated in MAWD/MAWBP-overexpressing cells. N-cadherin and Snail expression was strongr in vector-expressing cells and comparatively weaker in MAWD/MAWBP co-overexpressing cells. MAWD/MAWBP co-overexpression inhibited Smad2 phosphorylation and nuclear translocation (P < 0.05), and AKP activity was lowest in MAWD/MAWBP coexpressing cells and highest in vector-expressing cells (P < 0.001). TGF-beta, E-cadherin, and PGC expression in xenograft tumors derived from MAWD/MAWBP coexpressing cells was higher than that in control.ConclusionsMAWD and MAWBP were downregulated and associated with the differentiation grade in GC tissues. MAWD and MAWBP might induce the expression of differentiation-related proteins by modulating TGF-beta signaling in GC cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1637-7) contains supplementary material, which is available to authorized users.

Gene Expression Analysis of Urine Sediment: Evaluation for Potential Noninvasive Markers of Interstitial Cystitis/Bladder Pain Syndrome

We determined whether gene expression profiles in urine sediment could provide noninvasive markers for interstitial cystitis/bladder pain syndrome with and/or without Hunner lesions. Fresh catheterized urine was collected and centrifuged from 5 controls, and 5 Hunner lesion-free and 5 Hunner lesion bearing patients. RNA was extracted from pelleted material and quantified by gene expression microarray using the GeneChip® Human Gene ST Array. Three biologically likely hypotheses were tested, including 1) all 3 groups are distinct from each other, 2) controls are distinct from the 2 types combined of patients with interstitial cystitis/bladder pain syndrome and 3) patients with Hunner lesion-interstitial cystitis/bladder pain syndrome are distinct from controls and patients with nonHunner-lesion interstitial cystitis/bladder pain syndrome combined. For statistical parity an unlikely fourth hypothesis was included, that is patients with nonHunner-lesion interstitial cystitis/bladder pain syndrome are distinct from controls and patients with Hunner lesion-interstitial cystitis/bladder pain syndrome combined. Analysis supported selective up-regulation of genes in the Hunner lesion interstitial cystitis/bladder pain syndrome group (hypothesis 3), which were primarily associated with inflammation. The inflammatory profile was statistically similar to that reported in a prior Hunner lesion interstitial cystitis/bladder pain syndrome bladder biopsy study. Gene expression analysis of urine sediment was feasible in this pilot study. Expression profiles failed to discriminate nonHunner-lesion interstitial cystitis/bladder pain syndrome from controls and they are unlikely to be a noninvasive marker for nonHunner-lesion interstitial cystitis/bladder pain syndrome. In contrast, patients with Hunner lesion had increased proinflammatory gene expression in urine sediment, similar to that in a prior microarray study of bladder biopsies. If these preliminary results are validated in future research, they may lead to a noninvasive biomarker for Hunner lesion-interstitial cystitis/bladder pain syndrome.

PPARδ Activation Promotes Stratum Corneum Formation and Epidermal Permeability Barrier Development during Late Gestation

The goal of epidermal ontogenesis is to form a stratum corneum (SC), which is required for post-natal permeability barrier function. The regulation of epidermal ontogenesis is poorly understood, but nuclear hormone receptors have been shown to have an important function. As peroxisome proliferator-activated receptor-delta (PPARdelta) is very abundant in fetal epidermis and PPARdelta activation stimulates differentiation and permeability barrier formation in adults, we hypothesized that PPARdelta might regulate epidermal ontogenesis. Treatment of fetal rat explants with the PPARdelta ligand, GW 610742X, accelerates permeability barrier development, evidenced by a decrease in transepidermal water loss and an enhanced outside-in barrier function, attributable to the presence of more mature lamellar membranes in the SC and enhanced expression of loricrin and involucrin. Similarly, the intra-amniotic administration of GW 610742X also accelerates the formation of the SC and permeability barrier development. Finally, in PPARdelta-deficient mice the formation of the SC and the expression of differentiation-related proteins were delayed on days 16.5 and 17.5 of gestation. However, at later stages (day 18.5 and after birth), there were no differences between wild-type- and PPARdelta-deficient mice, indicating only a transient delay in epidermal ontogenesis. These studies show that PPARdelta has a role in SC formation and permeability barrier development.

Differentiation Associated Changes in Gene Expression Profiles of Interstitial Cystitis and Control Urothelial Cells

We evaluated gene expression profiles after inducing differentiation in cultured interstitial cystitis and control urothelial cells. Bladder biopsies were taken from patients with interstitial cystitis and controls, that is women undergoing surgery for stress incontinence. Primary cultures were grown in keratinocyte growth medium with supplements. To induce differentiation in some plates the medium was changed to Dulbecco's modified Eagle's minimal essential medium-F12 (Media Tech, Herndon, Virginia) with supplements. RNA was analyzed with Affymetrix(R) chips. Three patients with nonulcerative interstitial cystitis were compared with 3 controls. After inducing differentiation 302 genes with a described function were altered at least 3-fold in interstitial cystitis and control cells (p <0.01). Functions of the 162 up-regulated genes included cell adhesion (eg claudins, occludin and cingulin), urothelial differentiation, the retinoic acid pathway and keratinocyte differentiation (eg skin cornified envelope components). The 140 down-regulated transcripts included genes associated with basal urothelium (eg p63, integrins beta4, alpha5 and alpha6, basonuclin 1 and extracellular matrix components), vimentin, metallothioneins, and members of the Wnt and Notch pathways. When comparing interstitial cystitis control cells after differentiation, only 7 genes with a described function were altered at least 3-fold (p <0.01). PI3, SERPINB4, CYP2C8, EFEMP2 and SEPP1 were decreased, and AKR1C2 and MKNK1 were increased in interstitial cystitis cases. Differentiation associated changes occurred in interstitial cystitis and control cells. Comparing interstitial cystitis vs control cases revealed few differences. This study may have included patients with interstitial cystitis and minimal urothelial deficiency, and/or we may have selected cells that were most robust in culture. Also, the abnormal urothelium in interstitial cystitis cases may be due to post-translational changes and/or to the bladder environment.

Dynamic regulation of the human dopachrome tautomerase promoter by MITF, ER‐α and chromatin remodelers during proliferation and senescence of human melanocytes

Senescent cells are known to display altered gene expression of differentiation-associated genes. We have previously demonstrated that the melanocyte transcriptional regulator microphthalmia-associated protein (MITF) is down-regulated in senescent melanocytes. Since virtually nothing is known regarding the differentiated function of senescent melanocytes, we analyzed the transcriptional regulation of Dopachrome tautomerase (DCT), a member of the tyrosinase gene family, in proliferating and in senescent human melanocytes. Computational analysis of the region containing the M-box that includes the MITF CATGTG binding motif demonstrated that this sequence overlaps with the estrogen receptor alpha (ER-alpha), USF-1, TFE-3, Isl-1 and AP-1 binding elements. Electrophoresis gel-shift analysis using an oligonucleotide containing MITF and ERE elements identified MITF and ER-alpha complexes in proliferating melanocytes, whereas only ER-alpha complexes were detected in senescent cells. Importantly, a promoter-reporter analysis demonstrated that the coactivator p300/CBP switched MITF from a repressor to an activator of DCT transcription. p300/CBP was also required by ER-alpha and MITF to induce high, synergistic activation of the DCT promoter. We have also found that transcription of the DCT gene is differentially regulated by major melanocyte mitogens. In contrast to the activating effect of cAMP inducers, 12-O-tetradecanoylphorbolacetate (TPA) was a potent repressor of DCT transcription, suggesting that this gene can be differentially regulated by multiple environmental signals and promoter context. In support of this conclusion, trichostatin A, a histone deacetylase inhibitor, counteracted the TPA-mediated repression, and restored high levels of DCT protein in cultured melanocytes. We conclude that senescent melanocytes display dramatic changes in the expression of differentiation-related proteins; such changes may in turn result in altered melanocyte function and survival to environmental stresses.

The origin of vimentin expression in invasive breast cancer: epithelial–mesenchymal transition, myoepithelial histogenesis or histogenesis from progenitor cells with bilinear differentiation potential?

Vimentin expression is a rather rare finding in invasive breast cancer, and is associated with high tumour invasiveness and chemoresistance. It is currently explained by two different biological theories: direct histogenetic derivation from myoepithelial cells, and epithelial-mesenchymal transition (EMT) reflecting the end-stage of breast cancer dedifferentiation. In this study we aimed to obtain further insights into the biological hallmarks of these vimentin-expressing breast cancers. We applied immunohistochemistry for vimentin and 15 other differentiation markers to a series of 364 invasive breast cancer cases, using tissue microarray technology. 7.7% of all tumours expressed vimentin. Almost all of these cases (19/21) were Grade 3 invasive ductal carcinomas, and the majority (13/21) of these were associated with a ductal in situ component. Vimentin expression was also seen in the respective in situ components and correlated positively with the expression of SMA, CD10, CK 5, p53, Mib-1 and EGFR. A negative correlation was seen for the expression of CK 8/18 and the oestrogen receptor. Vimentin-expressing carcinomas revealed a significantly higher average absolute number of cytogenetic alterations per case, but a significantly lower frequency of chromosome 16q losses compared to vimentin-negative cases. Our present results demonstrate that, despite analogies between vimentin-positive breast cancers and myoepithelial cells in their expression of differentiation-related proteins, neither myoepithelial histogenesis nor EMT can exclusively explain the biology of these distinct tumours. This is mainly supported by the significantly higher incidence of vimentin-expressing breast cancers compared to any other myoepithelial breast tumours and the fact that vimentin is already observed in ductal in situ components. We therefore propose the alternative hypothesis that vimentin-expressing breast carcinomas may derive from breast progenitor cells with bilinear (glandular and myoepithelial) differentiation potential.

Expression of desmosomal proteins in rat keratinocytes during in vitro differentiation.

The keratinocyte, the major component of the epidermis, expresses several proteins that characterize the keratinization during the differentiation. Proliferation and differentiation of cultured human keratinocytes are known to be regulated by the Ca2+ concentration in the culture medium. However, informations about the rat keratinocyte are relatively limited and their physiology is still an open question. To elucidate the characteristics of the rat keratinocyte, we established rat keratinocyte culture system and examined effects of extracellular calcium concentration on the expression of differentiation-related proteins. Keratinocytes were isolated from the newborn rat skin with 0.25% trypsin, followed by separation with a Percoll density gradient. The separated cells were grown in MCDB 153 medium containing several growth factors and Ca(2+)-free fetal bovine serum, then stimulated with Ca2+. Immunoblotting demonstrated strong expression of beta1 integrin in unstimulated cells, suggesting that the primary culture of rat keratinocytes was successfully established. Expression of desmoglein and transglutaminase was increased by Ca2+ stimulation, whereas beta1 integrin expression was decreased in response to increasing concentrations of Ca2+. These observations indicate that cultured rat keratinocytes maintain the ability to differentiate in vitro, which is similar to that of the basal keratinocytes in the epidermis.