Hypoxia and hyperoxia differentially control proliferation of rat neural crest stem cells via distinct regulatory pathways of the HIF1α-CXCR4 and TP53-TPM1 proteins.

BACKGROUND: Neural crest stem cells (NCSCs) are a population of adult multipotent stem cells. We are interested in studying whether oxygen tensions affect the capability of NCSCs to self-renew and repair damaged tissues. NCSCs extracted from the hair follicle bulge region of the rat whisker pad were cultured in vitro under different oxygen tensions. RESULTS: We found significantly increased and decreased rates of cell proliferation in rat NCSCs (rNCSCs) cultured, respectively, at 0.5% and 80% oxygen levels. At 0.5% oxygen, the expression of both hypoxia-inducible factor (HIF) 1α and CXCR4 was greatly enhanced in the rNCSC nuclei and was suppressed by incubation with the CXCR4-specific antagonist AMD3100. In addition, the rate of cell apoptosis in the rNCSCs cultured at 80% oxygen was dramatically increased, associated with increased nuclear expression of TP53, decreased cytoplasmic expression of TPM1 (tropomyosin-1), and increased nuclear-to-cytoplasmic translocation of S100A2. Incubation of rNCSCs with the antioxidant N-acetylcysteine (NAC) overcame the inhibitory effect of 80% oxygen on proliferation and survival of rNCSCs. CONCLUSIONS: Our results show for the first time that extreme oxygen tensions directly control NCSC proliferation differentially via distinct regulatory pathways of proteins, with hypoxia via the HIF1α-CXCR4 pathway and hyperoxia via the TP53-TPM1 pathway. Developmental Dynamics 246:162-185, 2017. © 2016 Wiley Periodicals, Inc.

Simulated Microgravity Disrupts Cytoskeleton Organization and Increases Apoptosis of Rat Neural Crest Stem Cells Via Upregulating CXCR4 Expression and RhoA-ROCK1-p38 MAPK-p53 Signaling.

Neural crest stem cells (NCSCs) are a population of multipotent stem cells that are distributed broadly in many tissues and organs and are capable of differentiating into a variety of cell types that are dispersed throughout three germ layers. We are interested in studying the effects of simulated microgravity on the survival and self-renewal of NCSCs. NCSCs extracted from the hair follicle bulge region of the rat whisker pad were cultured in vitro, respectively, in a 2D adherent environment and a 3D suspension environment using the rotatory cell culture system (RCCS) to simulate microgravity. We found that rat NCSCs (rNCSCs) cultured in the RCCS for 24 h showed disrupted organization of filamentous actin, increased globular actin level, formation of plasma membrane blebbing and neurite-like artifact, as well as decreased levels of cortactin and vimentin. Interestingly, ∼70% of RCCS-cultured rNCSCs co-expressed cleaved (active) caspase-3 and neuronal markers microtubule-associated protein 2 (MAP2) and Tuj1 instead of NCSC markers, suggesting stress-induced formation of neurite-like artifact in rNCSCs. In addition, rNCSCs showed increased C-X-C chemokine receptor 4 (CXCR4) expression, RhoA GTPase activation, Rho-associated kinase 1 (ROCK1) and p38 mitogen-activated protein kinase (MAPK) phosphorylation, and p53 expression in the nucleus. Incubation of rNCSCs with the Gα protein inhibitor pertussis toxin or CXCR4 siRNA during RCCS-culturing prevented cytoskeleton disorganization and plasma membrane blebbing, and it suppressed apoptosis of rNCSCs. Taken together, we demonstrate for the first time that simulated microgravity disrupts cytoskeleton organization and increases apoptosis of rNCSCs via upregulating CXCR4 expression and the RhoA-ROCK1-p38 MAPK-p53 signaling pathway.

Analysis of dermal papilla cell interactome using STRING database to profile the ex vivo hair growth inhibition effect of a vinca alkaloid drug, colchicine.

Dermal papillae (DPs) control the formation of hair shafts. In clinical settings, colchicine (CLC) induces patients' hair shedding. Compared to the control, the ex vivo hair fiber elongation of organ cultured vibrissa hair follicles (HFs) declined significantly after seven days of CLC treatment. The cultured DP cells (DPCs) were used as the experimental model to study the influence of CLC on the protein dynamics of DPs. CLC could alter the morphology and down-regulate the expression of alkaline phosphatase (ALP), the marker of DPC activity, and induce IκBα phosphorylation of DPCs. The proteomic results showed that CLC modulated the expression patterns (fold>2) of 24 identified proteins, seven down-regulated and 17 up-regulated. Most of these proteins were presumably associated with protein turnover, metabolism, structure and signal transduction. Protein-protein interactions (PPI) among these proteins, established by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, revealed that they participate in protein metabolic process, translation, and energy production. Furthermore, ubiquitin C (UbC) was predicted to be the controlling hub, suggesting the involvement of ubiquitin-proteasome system in modulating the pathogenic effect of CLC on DPC.

Interactomics profiling of the negative regulatory function of carbon monoxide on RANKL-treated RAW 264.7 cells during osteoclastogenesis.

BACKGROUND: During osteoclastogenesis, the maturation of osteoclast (OC) progenitors is stimulated by the receptor activator of nuclear factor-κB ligand (RANKL). Excess OC production plays a critical role in the pathogenesis of inflammatory bone disorders. Conversely, the inhibition of abnormal OC proliferation reduces inflammation-induced bone loss. Low concentrations of carbon monoxide (CO) are known to decrease inflammation and OC-mediated bone erosion but the molecular mechanism is unknown. RESULTS: To obtain insight into the biological function of CO, cultured RANKL-treated RAW 264.7 cells were used in an in vitro experimental model of osteoclastogenesis. The results showed that CO inhibited: 1) tartrate-resistant acid phosphatase (TRAP)-positive cell formation; 2) F-actin ring production; 3) c-fos pathway activation; 4) the expression of cathepsin K, TRAP, calcitonin receptor, and matrix metalloproteinase-9 mRNAs; 5) the expression of nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 in translation. Protein-protein interaction analysis predicted mitogen-activated protein kinase kinase kinase 4 as the controlling hub. CONCLUSIONS: Low-concentrations of CO (250 ppm) may inhibit osteoclastogenesis. Data from STRING- and IPA-based interactome analyses suggested that the expression of proteins with the functions of signal transduction, enzymes, and epigenetic regulation are significantly altered by CO during RANKL-induced osteoclastogenesis. Our study provides the first interactome analysis of osteoclastogenesis, the results of which supported the negative regulation of OC differentiation by CO.

The total synthesis of a ganglioside Hp-s1 analogue possessing neuritogenic activity by chemoselective activation glycosylation.

The total synthesis of ganglioside 2, an analogue of the ganglioside Hp-s1 (1) which displays neuritogenic activity toward the rat pheochromocytoma cell line PC-12 cell in the presence of nerve growth factor (NGF) with an effect (34.0%) greater than that of the mammalian ganglioside GM 1 (25.4%), was accomplished by applying a chemoselective-activation glycosylation strategy. Moreover, we also demonstrate that the synthesized ganglioside 2 exhibited neuritogenic activity toward the human neuroblastoma cell line SH-SY5Y without the presence of NGF.

Proteomics and bioinformatics analysis of lovastatin-induced differentiation in ARO cells.

Lovastatin (lova), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, can induce differentiation in cancer cells at low concentration, thus having potential to be used as an auxiliary agent in cancer therapy. However, biological networks associated with the differentiation effect of lova have not been elucidated. To investigate molecular mechanisms of lova, the present study was aimed at proteomics and bioinformatics analyses on anaplastic thyroid cancer cell line ARO differentiated with low concentration of lova. Thyroid differentiation was induced by treating ARO cells with 25 µM of lova and confirmed by checking upregulation of some thyroid differentiation markers. Gel-based proteomics analysis was then performed to identify proteins differentially expressed between undifferentiated and lova-differentiated ARO cells. Bioinformatics analysis was finally performed to estimate biological networks regulated by lova. Our results showed that lova impacted on proteins involved in protein folding, biomolecule metabolism, signal transduction, protein expression and protein degradation. Specifically, transfecting ARO cells with plasmid DNA encoding flotillin 1 (FLOT1) up-regulated the thyroid differentiation markers, indicating that FLOT1 might at least partially mediate the lova-induced thyroid differentiation. These data may shed light on the mechanism underlying lova-induced re-differentiation of thyroid cancer, and give a rationale for clinical use of lova as an auxiliary agent in cancer therapy.

Proteomic analysis and identification of aqueous humor proteins with a pathophysiological role in diabetic retinopathy.

Diabetic retinopathy (DR) can cause irreversible blindness and is the severest microvascular complication in the eyes of patients with diabetic mellitus (DM). The identification of susceptibility factors contributing to development of DR is helpful for identifying predisposed patients and improving treatment efficacy. Although proteomics analysis is useful for identifying protein markers related to diseases, it has never been used to explore DR-associated susceptibility factors in the aqueous humor (AH). To better understand the pathophysiology of DR and to identify DR-associated risk factors, a gel-based proteomics analysis was performed to compare AH protein profiles of DM patients with and without development of DR. MALDI-TOF MS was then performed to identify protein spots that were differentially expressed between the two groups and western blot analysis was used to validate the expressional change of protein demonstrated by proteomics. Our proteomics and bioinformatics analysis identified 11 proteins differentially expressed between DR and control groups. These proteins are linked to biological networks associated with nutrition transport, microstructure reorganization, angiogenesis, anti-oxidation, and neuroprotection. The data may provide potential AH biomarkers and susceptibility factors for predicting DR development, and provide an insight into the underlying pathophysiological mechanisms of DR. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Impact of taxol on dermal papilla cells--a proteomics and bioinformatics analysis.

Dermal papilla (DP) cells play a regulatory role in hair growth, and also play a role in alopecia (hair loss). However, effects of taxol, which is a widely used chemotherapy drug, on DP cells remain unclear, despite that theoretically taxol can impact on DP cells to contribute to taxol-induced alopecia. To better understand pathophysiology of taxol-induced damage in DP cells, morphological and biochemical analyses were performed to check whether taxol can cause apoptosis in cultured DP cells or not. If it can, proteomics and bioinformatics analyses were then performed to investigate the protein networks which are impacted by the taxol treatment. Our data showed that taxol can cause apoptotic damage in DP cells in a concentration-dependant manner, as demonstrated by various apoptotic markers. Proteomic analysis on DP cells treated with the lowest apoptosis-inducible concentration of taxol revealed that taxol can affect expression of proteins involved in Ca2+-regulated biological processes, vesicles transport, protein folding, reductive detoxification, and biomolecules metabolism. Furthermore, bioinformatics analysis indicated that taxol can impact on multiple biological networks. Taken together, this biochemical, proteomics, and bioinformatics data may give an insight into pathophysiology of taxol-induced damage in DP cells and shed light on mechanisms underlying taxol-induced alopecia.

Proteomics demonstration that histone H4 is a colchicine-induced retro-modulator of growth and alkaline phosphatase activity in hair follicle dermal papilla culture.

Dermal papilla cells (DPCs) control the development of hair follicles via cell-cell interactions and extracellular molecules. Colchicine affected active anagen DPCs to result in hair loss in the clinical setting. The purpose of this study was to identify the retro-modulator released by DPCs exposed to sub-toxic dose of colchicine and elucidate its effect on dermal papilla culture. The molecular-weight cutoff ultrafiltration and HPLC were used to purify the components of colchicine-treated DPC secretomes and examined their ability to down-regulate the growth and alkaline phosphatase (ALP) activity of DPCs. The active product was identified by in-gel trypsin digestion, nano-LC-ESI-MS/MS and validated by Western blot to be histone H4 (P62804), which inhibited the proliferation and diminished the ALP activity of cultured DPCs. Treating DPCs with recombinant histone H4 reproduced the growth inhibition effect whereas adding antibody to immunoneutralize histone H4 abolished this growth inhibitory consequence. DPCs with high ALP activity can induce the neogenesis of hair follicles and support the hair fiber growth in vivo. Our results indicated that sub-lethal colchicine can inactivate DPCs through releasing histone H4. Through the investigation of the retro-modulation of histone H4 on dermal papillae may give implications for understanding the mechanism of colchicine-induced hair disorder.