Transcription Profiles Reveal Age-Dependent Variations of Photosynthetic Properties and Sugar Metabolism in Grape Leaves (Vitis vinifera L.).

Leaves, considered as the 'source' organs, depend on the development stages because of the age-dependent photosynthesis and assimilation of leaves. However, the molecular mechanisms of age-dependent limitations on the function of leaves are seldom reported. In the present study, the photosynthesis-related characteristics and photoassimilates were investigated in grape leaves at six different age groups (Ll to L6) at micro-morphological, biochemical, and molecular levels. These results showed lower expression levels of genes associated with stomatal development, and chl biosynthesis resulted in fewer stomata and lowered chlorophyll a/b contents in L1 when compared to L3 and L5. The DEGs between L5 and L3/L1 were largely distributed at stomatal movement, carbon fixation, and sucrose and starch metabolism pathways, such as STOMATAL ANION CHANNEL PROTEIN 1 (SLAC1), FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE (FBA1), SUCROSE-PHOSPHATE SYNTHASE (SPP1), and SUCROSE-PHOSPHATE PHOSPHATASE (SPS2, 4). These genes could be major candidate genes leading to increased photosynthesis capacity and sugar content in L5. The accumulation of starch grains in the chloroplast and palisade tissue of L5 and higher transcription levels of genes related to starch biosynthesis in L5 further supported the high ability of L5 to produce photoassimilates. Hence, our results provide insights for understanding different photosynthetic functions in age-dependent leaves in grape plants at the molecular level.

Genome-Wide Identification and Expression Analysis of Senescence-Associated Genes in Grapevine (Vitis vinifera L.) Reveal Their Potential Functions in Leaf Senescence Order.

Natural leaf senescence is an acclimation strategy that enables plants to reallocate nutrients. In the present study, interestingly, we found that the basal mature leaves of grapevine primary shoots (P) exhibited the earliest senescence, followed by the apical young leaves of secondary shoots (ST), and then the basal mature leaves of secondary shoots (S). The Chl level decreased with the extent of leaf senescence. According to the genome-wide identification and expression analysis, sixteen senescence-associated genes (SAGs) involved in Chl breakdown were identified in the grapevine genome. Their expression patterns showed that the transcript changes in VvSGR, VvPPH2, and VvFtsH6-2 corresponded to the changes in Chl content among P, S, and ST. The changes in the transcription of VvNYC1, VvSGR, VvPAO1, VvPAO2, VvPAO4, VvPPH1, VvPPH3, and VvFtsH6-1 only contributed to low Chl levels in P. The cis-element analysis indicated that these SAGs possessed several light- and hormone-responsive elements in their promoters. Among them, ABA-responsive elements were found in twelve of the sixteen promoters of SAGs. Correspondingly, ABA-signaling components presented various changes in transcription among P, S, and ST. The transcription changes in VvbZIP45 and VvSnRK2.1 were similar to those in VvSGR, VvPPH2, and VvFtsH6-2. The other nine ABA-signaling components, which included VvRCAR2, VvRCAR4, VvRCAR6, VvRCAR7, VvRCAR2, VvPP2C4, VvPP2C9, VvbZIP25, and VvSnRK2.3, were highly expressed in P but there was no difference between S and ST, with similar expression patterns for VvNYC1, VvSGR, VvPAO1, VvPAO2, VvPAO4, VvPPH1, VvPPH3, and VvFtsH6-1. These results suggested that the senescence of P and ST could be regulated by different members of Chl breakdown-related SAGs and ABA-signaling components. These findings provide us with important candidate genes to further study the regulation mechanism of leaf senescence order in grapevine.

CAFs-derived MFAP5 promotes bladder cancer malignant behavior through NOTCH2/HEY1 signaling.

Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment and contribute to tumor cell proliferation and metastasis. Microfibrillar-associated protein 5 (MFAP5), a component of elastic microfibers and an oncogenic protein in several types of tumors, is secreted by CAFs. However, the role of MFAP5 in the bladder cancer remains unclear. Here, we report that MFAP5 is upregulated in bladder cancer and is associated with poor patient survival. Downregulation of MFAP5 in CAFs led to an impairment in proliferation and invasion of bladder cancer cells. Luciferase reporter assays and electrophoretic mobility shift assays (EMSA) showed QKI directly downregulates MFAP5 in CAFs. In addition, CAFs-derived MFAP5 led to an activation of the NOTCH2/HEY1 signaling pathway through direct interaction with the NOTCH2 receptor, thereby stimulating the N2ICD release. RNA-sequencing revealed that MFAP5-mediated PI3K-AKT signaling activated the DLL4/NOTCH2 pathway axis in bladder cancer. Moreover, downregulation of NOTCH2 by short hairpin RNA or the inactivating anti-body NRR2Mab was able to reverse the adverse effects of MFAP5 stimulation in vitro and in vivo. Together, these results demonstrate CAFs-derived MFAP5 promotes the bladder cancer proliferation and metastasis and provides new insight for targeting CAFs as novel diagnostic and therapeutic strategy.

Heat injured stromal cells-derived exosomal EGFR enhances prostatic wound healing after thulium laser resection through EMT and NF-κB signaling.

BACKGROUND: This study investigated shallow heat injury to prostate stromal fibroblasts and epithelial cells and their interaction to regulate the wound healing and the underlying molecular events. METHODS: Prostate stromal fibroblasts and epithelial cells were cultured individually or cocultured and subjected to shallow heat injury for assessments of cell proliferation, migration, apoptosis, cell cycle distribution, and gene expression. The supernatant of heat-injured WPMY-1 cells was collected for exosome extraction and assessments. Furthermore, beagle dogs received thulium laser resection of the prostate (TmLRP) and randomly divided into Gefitinib, GW4869, and control treatment for the histological analysis, tissue re-epithelialization, and epidermal growth factor receptor (EGFR) expression on the prostatic wound surface. Immunofluorescence was to evaluate p63-positive basal progenitor cell trans-differentiation and macrophage polarization and ELISA was to detect cytokine levels in beagles' urine. RESULTS: Shallow heat injury caused these cells to enter a stressed state and enhanced their crosstalk. The prostate stromal fibroblasts produced and secreted more exosomal-EGFR and other cytokines and chemokines after shallow heat injury, resulting in increased proliferation and migration of prostate epithelial cells during wound healing. The wound healing of the canine prostatic urethra following the TmLRP procedure was slower in the Gefitinib and GW4869 treatment group than in the control group of animals. Immunofluorescence and ELISA showed that reduced EGFR expression interrupted macrophage polarization but increased the inflammatory response. CONCLUSIONS: Shallow heat injury was able to promote the interaction of prostate stromal cells with prostate epithelial cells to enhance wound healing. Stromal-derived exosomal-EGFR plays a crucial role in the balance of the macrophage polarization and prostatic wound healing.

M1 macrophage mediated increased reactive oxygen species (ROS) influence wound healing via the MAPK signaling in vitro and in vivo.

Thulium laser resection of the prostate (TmLRP), a major treatment for benign prostatic hyperplasia (BPH), has several postoperative complications that affect the patients' quality of life. The aim of this study was to investigate the effect of the M1 macrophage-secreted reactive oxygen species (ROS) on prostatic wound healing, and the role of MAPK signaling in this process. A co-culture model in vitro was established using macrophages and prostate epithelial or stromal cells. Cell proliferation, migration, apoptosis, MAPK pathway-related gene expression levels were evaluated by standard assays. In addition, an in vivo model of prostatectomy was established in beagles by subjecting them to TmLRP, and were either treated with N-acetyl-L-cysteine (NAC) and or placebo. Wound healing and re-epithelialization were analyzed histopathologically in both groups, in addition to macrophage polarization, oxidative stress levels and MAPK pathway-related proteins expressions. Intracellular ROS levels were significantly increased in the prostate epithelial and stromal cells following co-culture with M1-like macrophages and H2O2 exposure via MAPK activation, which affected their proliferation, migration and apoptosis, and delayed the wound healing process. The cellular functions and wound healing capacity of the prostate cells were restored by blocking or clearing the macrophage-secreted ROS. In the beagle model, increased ROS levels impaired cellular functions, and appropriate removing ROS accelerated the wound healing process.

Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway.

Background: M2 macrophages are well accepted to promote cancer progression in the prostate cancer (PCa). Paracrine is the principally studied mode of communication between M2 macrophages and tumor cells. In addition to this, we present here a novel model to demonstrate these cellular communications. Methods: PCa cells were co-cultured with THP-1/ human peripheral blood mononuclear cells derived M2 macrophages in direct contact manner. Cancer cell proliferation and invasion were examined to explain how direct contact communicates. Cell-based findings were validated in two xenograft models and patients samples. Results: M2 macrophage direct contact induced a higher proliferation and invasion in PCa cells when compared with noncontact coculture manner. In direct contact manner, NOTCH1 pathway was greatly activated in PCa cells, induced by elevated γ-secretase activity and higher coactivator MAML2 expression. Additionally, blocking γ-secretase activity and depletion of MAML2 completely abolished M2 macrophage direct contact-mediated PCa cell proliferation and invasion. In vivo, inhibiting NOTCH1 signalling impaired M2 macrophage-mediated PCa tumor growth and lung metastasis. Notably, M2 macrophage infiltration as well as high NOTCH1 signaling in cancer cells indicated more aggressive features and worse survival in PCa patients. Conclusion: Our results demonstrated the cell-cell direct contact pattern is an important way in PCa microenvironment cell communication. In this manner, elevated γ-secretase activity and MAML2 expression induced higher NOTCH1 signalling in PCa cells, which increased tumor cells proliferation and invasion. This potentially provided a therapeutic target for PCa.