Adrenomedullin promotes human endothelial cell proliferation via HIF-1α

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.     

SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-β1 activity

Pericytes migrate to nascent vessels and promote vessel stability. Recently, we reported that secreted protein acidic and rich in cysteine (SPARC)-deficient mice exhibited decreased pericyte-associated vessels in an orthotopic model of pancreatic cancer, suggesting that SPARC influences pericyte behavior. In this paper, we report that SPARC promotes pericyte migration by regulating the function of endoglin, a TGF-β1 accessory receptor. Primary SPARC-deficient pericytes exhibited increased basal TGF-β1 activity and decreased cell migration, an effect blocked by inhibiting TGF-β1. Furthermore, TGF-β-mediated inhibition of pericyte migration was dependent on endoglin and αV integrin. SPARC interacted directly with endoglin and reduced endoglin interaction with αV integrin. SPARC deficiency resulted in endoglin-mediated blockade of pericyte migration, aberrant association of endoglin in focal complexes, an increase in αV integrins present in endoglin immunoprecipitates, and enhanced αV integrin-mediated activation of TGF-β. These results demonstrate that SPARC promotes pericyte migration by diminishing TGF-β activity and identify a novel function for endoglin in controlling pericyte behavior.     

PP1α, PP1β and Wip-1 regulate H4S47 phosphorylation and deposition of histone H3 variant H3.3

Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo, whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo. These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells.     

HIF-1α promotes the proliferation and migration of pulmonary arterial smooth muscle cells via activation of Cx43

The proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important cause of pulmonary vascular remodelling in hypoxia-induced pulmonary hypertension (HPH). However, its underlying mechanism has not been well elucidated. Connexin 43 (Cx43) plays crucial roles in vascular smooth muscle cell proliferation in various cardiovascular diseases. Here, the male Sprague-Dawley (SD) rats were exposed to hypoxia (10% O₂) for 21 days to induce rat HPH model. PASMCs were treated with CoCl₂ (200 µM) for 24 h to establish the HPH cell model. It was found that hypoxia up-regulated the expression of Cx43 and phosphorylation of Cx43 at Ser 368 in rat pulmonary arteries and PASMCs, and stimulated the proliferation and migration of PASMCs. HIF-1α inhibitor echinomycin attenuated the CoCl₂-induced Cx43 expression and phosphorylation of Cx43 at Ser 368 in PASMCs. The interaction between HIF-1α and Cx43 promotor was also identified using chromatin immunoprecipitation assay. Moreover, Cx43 specific blocker (^37,43Gap27) or knockdown of Cx43 efficiently alleviated the proliferation and migration of PASMCs under chemically induced hypoxia. Therefore, the results above suggest that HIF-1α, as an upstream regulator, promotes the expression of Cx43, and the HIF-1α/Cx43 axis regulates the proliferation and migration of PASMCs in HPH.     

MMP-11 promotes papillary thyroid cell proliferation and invasion via the NF-κB pathway

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer, and its incidence is on the rise. It has been reported that some matrix metalloproteinases (MMPs) are abnormally expressed in PTC and can be used as diagnostic markers. However, few studies have explored the underlying mechanisms by which MMPs promote tumor progression. In this study, we used microarray analysis to compare the variations of gene expression within the PTC cell populations and their adjacent normal tissues and found that MMP-11 was the most differentially expressed MMP. To investigate the role of MMP-11 in the mediation of thyroid cancer cell development, pEnter-MMP-11 plasmid, and MMP-11 small interfering RNA were applied to up- and downregulate MMP-11 expression of in cultured PTC cell lines K1 and BCPAP. The results suggested that the levels of proliferation and migration of cells transfected with MMP-11 siRNA were significantly reduced, while the levels in MMP-11-plasmid-transfected cells were increased. In terms of the mechanism, experimental data showed that the change in cyclin D1 is consistent with MMP-11 expression, which may explain the changes in proliferation. In addition, Western blot assay was conducted to analyze the p65 and activated (phospho-) p65 protein levels concomitant with MMP-11 adjustments. Variations in intracellular MMP-11 significantly altered the amount of phospho-p65 in thyroid cells, while p65 knockdown did not affect MMP-11 expression. These results suggest that MMP-11 is located upstream of p65 and regulates its activity. Interestingly, the data for the Transwell assay suggested that MMP-11 regulatory migration is also associated with the NF-κB p65 signaling pathway. In conclusion, this report describes the important role of MMP-11 in the regulation of thyroid cell proliferation and migration. Mechanistic studies have shown that cyclin D1 and p65 are important mediators in the processes, which provides a new way to study the mechanism of MMPs promoting the progression of thyroid cancer.     

TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation

Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF‐α plays an important role in the destruction of acinar structures. Here we examined TNF‐α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS‐SV‐AC) cells were treated with TNF‐α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF‐α treatment were investigated. TNF‐α‐treatment of NS‐SV‐AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS‐SV‐AC cells treated with TNF‐α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF‐κB activity in the TNF‐α‐induced suppression of AQP5 expression in NS‐SV‐AC cells, we detected similar TNF‐α suppression of AQP5 expression in non‐transfected cells and in a super‐repressor form of IκBα cDNA‐transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF‐α in NS‐SV‐AC cells. Therefore, our results may indicate that TNF‐α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.     

FAM29A promotes microtubule amplification via recruitment of the NEDD1–γ-tubulin complex to the mitotic spindle

Microtubules (MTs) are nucleated from centrosomes and chromatin. In addition, MTs can be generated from preexiting MTs in a γ-tubulin–dependent manner in yeast, plant, and Drosophila cells, although the underlying mechanism remains unknown. Here we show the spindle-associated protein FAM29A promotes MT-dependent MT amplification and is required for efficient chromosome congression and segregation in mammalian cells. Depletion of FAM29A reduces spindle MT density. FAM29A is not involved in the nucleation of MTs from centrosomes and chromatin, but is required for a subsequent increase in MT mass in cells released from nocodazole. FAM29A interacts with the NEDD1–γ-tubulin complex and recruits this complex to the spindle, which, in turn, promotes MT polymerization. FAM29A preferentially associates with kinetochore MTs and knockdown of FAM29A reduces the number of MTs in a kinetochore fiber, activates the spindle checkpoint, and delays the mitotic progression. Our study provides a biochemical mechanism for MT-dependent MT amplification and for the maturation of kinetochore fibers in mammalian cells.     

Chemical crosslinking of histone H1° to histone neighbours in nuclei and chromatin

Crosslinking of histones in mouse liver nuclei and extended chromatin with a bifunctional reagent leads to the formation of H1H1° heterodimers as well as H1°H1° homodimers. H1° can be also crosslinked to the core histones. Thus, the location of histone H1° within the basic repeating chromatin structure seems to be analogous to that of H1 histone.     

Retinoic acid promotes proliferation of chicken primordial germ cells via activation of PI3K/Akt-mediated NF-κB signalling cascade

As embryonic progenitors for the gametes, PGCs (primordial germ cells) proliferate and develop under strict regulation of numerous intrinsic and external factors. As the most active natural metabolite of vitamin A, all-trans RA (retinoic acid) plays pivotal roles in regulating development of various cells. The proliferating action of RA on PGCs was investigated along with the intracellular PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B; also known as Akt)-mediated NF-κB (nuclear factor κB) signalling cascade. The results show that RA significantly promoted PGC proliferation in a dose- and time-dependent manner, confirmed by BrdU (bromodeoxyuridine) incorporation and cell cycle analysis. However, this promoting effect was attenuated by sequential inhibitors of LY294002 for PI3K, KP372-1 for Akt and SN50 for NF-κB respectively. Western blot analysis showed increased Akt phosphorylation (Ser473) of PGCs after stimulation with RA, but this was abolished by LY294002 or KP372-1. Treatment with RA increased expression of NF-κB and decreased IκBα (inhibitory κBα) expression, which were inhibited by SN50. Blockade of PI3K or Akt activity inhibited NF-κB translocation from the cytoplasm to the nucleus. Finally, mRNA expression of cell cycle regulating genes [cyclin D1 and E, CDK6 (cyclin-dependent kinase 6) and CDK2] was up-regulated in the RA-treated cells. This stimulation was also markedly retarded by combined treatment with LY294002, KP372-1 and SN50. These results suggest that RA activates the PI3K/Akt and NF-κB signalling cascade to promote proliferation of the cultured chicken PGCs.     

TWEAK/Fn14 promotes the proliferation and collagen synthesis of rat cardiac fibroblasts via the NF-кB pathway

We wished to elucidate a potential role of the tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible molecule 14 (Fn14) axis in myocardial fibrosis. Stimulation of neonatal rat cardiac fibroblasts (CFs) with TWEAK could increase CFs numbers and collagen synthesis. Conversely, when CFs were pretreated with siRNA against Fn14, induction of cell proliferation and collagen synthesis by TWEAK were inhibited. Pretreatment with TWEAK on CFs induced activation of the nuclear factor-kappaB (NF-кB) pathway and subsequently increased the production of metalloproteinase-9 (MMP-9). Cell treatment with siRNA against Fn14 led to inhibition of the NF-кB pathway. Additionally, after stimulation of cell with ammonium pyrrolidine dithiocarbamate, cell proliferation and collagen synthesis induced by NF-кB and the upregulation of MMP-9 production were inhibited. The present study suggested that the TWEAK/Fn14 axis increased cell proliferation and collagen synthesis by activating the NF-кB pathway and increasing MMP-9 activity. This axis may be important for regulating myocardial fibrosis.