Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

Substance P preserves pancreatic ß-cells in type 1 and type 2 diabetic mice.

Preservation of pancreatic ß-cells is required for the development of therapies for type 1 and type 2 diabetes (T1D and T2D, respectively). Our previous study demonstrated that substance P (SP) preserves ß-cell populations in mice with streptozotocin-induced T1D. Here, we demonstrated that chronic systemic treatment with SP restored the mass of ß-cells both in nonobese diabetic (NOD) mice with T1D or db/db mice with T2D. SP delayed the onset of T1D in NOD mice via immune modulation. SP inhibited immune infiltration into islets and the salivary glands of NOD mice. In db/db mice, SP treatment rescued glucose intolerance. Moreover, SP inhibited apoptosis, as well as the activation of pancreatic stellate cells in pancreatic islets of db/db mice. SP downregulated the number of α-smooth muscle actin (α-SMA) expressing cells in db/db pancreatic islets. Cleaved-caspase-3 expression was reduced in islets of SP-treated db/db mice compared to that in the control. Therefore, these results suggested that SP may preserve pancreatic ß-cells through immune modulation and protection from the stimulated activation of pancreatic stellate cells and apoptosis in T1D and T2D, respectively.

Substance P preserves pancreatic ß-cells in streptozotocin-induced type 1 diabetic mice.

Preservation of the pancreatic ß-cell population is required for the development of therapies for diabetes, which is caused by a decrease in ß-cells. Here, we demonstrate the antidiabetic effects of substance P (SP) in type 1 diabetes (T1D) mice induced with streptozotocin. SP enhanced the compensatory proliferation of ß-cells in order to restore ß-cells in response to acute injury induced by a single high-dose of streptozotocin. However, SP affected neither the basal proliferation of ß-cells nor their apoptosis. In vitro studies by using the INS-1 pancreatic ß-cell line showed that SP mediated the increase in the proliferation of ß-cells via the activation of Akt. Chronic systemic treatment with SP restored the mass of ß-cells and inhibited insulitis in T1D mice induced with multiple low-doses of streptozotocin. Therefore, systemic treatment with SP may be a promising therapeutic strategy for treating diabetes in patients with loss of functional ß-cells.

Substance P accelerates wound healing in type 2 diabetic mice through endothelial progenitor cell mobilization and Yes-associated protein activation.

Wound healing is delayed in diabetes due to a number of factors, including impaired angiogenesis and poor dermal healing. The present study demonstrated that subcutaneous administration of substance P (SP) accelerates wound healing in db/db type 2 diabetic mice (db/db mice). SP injection (10 nM/kg, subcutaneously) enhanced angiogenesis, induced the mobilization of endothelial progenitor cells (EPCs) and increased the number of EPC­colony forming units (EPC­CFUs) in the bone marrow of db/db mice. Immunohistochemistry was performed to check the effects of SP on the cellular proliferation and the subcellular localization of Yes-associated protein (YAP) in the wound dermis. SP also upregulated cellular proliferation in the injured dermis of db/db mice. Compared with the control group, an increased number of cells in the wound dermis of SP-treated mice exhibited nuclear localization of YAP, which induces cellular proliferation. The results of the current study indicate that subcutaneous administration of SP may be a promising therapeutic strategy to treat diabetic wounds exhibiting impaired angiogenesis and dysfunctional dermal wound healing.

Reduced Expression of YAP in Dermal Fibroblasts is Associated with Impaired Wound Healing in Type 2 Diabetic Mice.

Dermal fibroblasts play essential roles in wound healing and their dysfunction has been shown to be associated with impaired wound healing in diabetes. In the present study, we aimed at investigating whether Yes-associated protein (YAP), a mediator of mechanotransduction in dermal fibroblasts, is associated with impaired wound healing in diabetic mice. Compared with that in the control, the rate of wound contraction was decreased twofold in db/db type 2 diabetic mice (db/db mice). To mimic diabetic pathological condition, dermal fibroblasts were cultured under high glucose conditions (25.5 mM glucose). Further, dermal fibroblast-mediated contraction of wound was evaluated by in vitro collagen gel contraction assay. Dermal fibroblasts cultured under hyperglycemic condition showed impaired gel contraction and mitochondrial dysfunction, compared to the cells cultured under normoglycemic conditions (5.5 mM glucose). Importantly, compared with the normal dermal fibroblasts, diabetic db/db dermal fibroblasts expressed lower levels of growth factors and cytokines that enhance wound healing, such as insulin-like growth factor-1, stromal cell-derived factor-1, connective tissue growth factor, and transforming growth factor-ß (TGF-ß). The quantity of YAP mRNA was also lower in diabetic db/db dermal fibroblasts, compared with that in the control fibroblasts. These results indicate that impaired wound healing in diabetics is associated with the dysfunction of dermal fibroblasts, including downregulation of YAP, which plays essential roles in extracellular matrix remodeling and TGF-ß-mediated wound healing.

Substance P enhances EPC mobilization for accelerated wound healing.

Wound healing is essential for the survival and tissue homeostasis of unicellular and multicellular organisms. The current study demonstrated that the neuropeptide substance P (SP) accelerated the wound healing process, particularly in the skin. Subcutaneous treatment of SP accelerated wound closing, increased the population of α-smooth muscle actin positive myofibroblasts, and increased extracellular matrix deposition at the wound site. Moreover, SP treatment enhances angiogenesis without a local increase in the expression levels of vascular endothelial growth factor and stromal cell-derived factor-1. Importantly, SP treatment increased both the population of circulating endothelial progenitor cells in the peripheral blood and in CD31 positive cells in Matrigel plugs. The tube forming potential of endothelial cells was also enhanced by SP treatment. The results suggested that the subcutaneous injection of SP accelerated the wound healing in the skin via better reconstitution of blood vessels, which possibly followed an increase in the systemic mobilization of endothelial progenitor cells and a more effective assembly of endothelial cells into tubes.

Substance P modulates properties of normal and diabetic dermal fibroblasts.

Dermal fibroblasts play essential roles in wound healing. However, they lose their normal regenerative functions under certain pathologic conditions such as in chronic diabetic wounds. Here, we show that substance P (SP) rescues the malfunctions of dermal fibroblasts in diabetes. SP increased the proliferation of diabetic dermal fibroblasts dose-dependently, although the effect was lower compared to the SP-stimulated proliferation of normal dermal fibroblasts. In contrast to normal dermal fibroblasts, SP increased the expression level of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) in diabetic dermal fibroblast hence, rescuing their angiogenic potential. The cellular characteristics of diabetic dermal fibroblasts modulated by SP would be able to accelerate the wound healing process through faster wound contraction and improved angiogenesis in diabetic chronic wounds. Moreover, SP pretreatment into dermal fibroblasts isolated from diabetic patients would be a promising strategy to develop autologous cell therapy for treating diabetic chronic wounds.

Substance P and thiorphan synergically enhance angiogenesis in wound healing.

Impaired angiogenesis is a common pathological characteristic of chronic wounds. Therefore, the regulation of angiogenesis is important for proper tissue repair. It was reported that substance P (SP) accelerates wound healing in a skin injury model. SP is degraded by neutral endopeptidase (NEP). Our study shows that systemic co-treatment of SP and thiorphan, an inhibitor of NEP synergically increased the number of α-smooth muscle actin positive-blood vessels in skin wounds. However, there was no synergic improvement in wound contraction and extracellular matrix deposition. Therefore, inhibition of endogenous NEP activity by thiorphan treatment might modulate the effects of SP treatment specifically on accelerating angiogenesis during wound healing. However, the molecular mechanism(s) of the synergic increase in angiogenesis by SP and thiorphan treatment is still unknown.

Effect of substance P on recovery from laser-induced retinal degeneration.

Retinal degeneration is caused by neovascularization and persistent inflammation in the retinal pigment epithelium (RPE) and choroid, and causes serious eye disease including age-related macular degeneration (AMD). Thus, inhibiting inflammation and neovascularization may be a primary approach to protect the retina from degeneration. The purpose of this study was to determine whether substance P (SP), which can suppress inflammation and mobilize stem cells, can protect the RPE from degeneration. The effect of SP was evaluated by analyzing systemic inflammation, cell survival, and neovascularization within the argon laser-injured retina of mice. At 1 week postinjury, the SP-treated group had lower tumor necrosis factor-alpha and higher interleukin-10 serum concentrations, and a more intact retinal structure compared to the vehicle-treated group. In mice administered SP repeatedly for 4 weeks, the retinal structure appeared normal and showed sparse neovascularization, whereas the vehicle-treated group showed severe retinal destruction and dense neovascularization. Moreover, the efficacy of SP was identical to that of mesenchymal stem cells that were transplanted into the vitreous after retinal injury. This study highlights the potential for the endogenous neuropeptide SP as a treatment for retinal damage to prevent conditions such as AMD.

Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper protein, regulates leaf growth by promoting cell expansion and endoreduplication.

Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper class I (HD-Zip I) gene, is highly expressed in leaves and stems, and induced by abiotic stresses, but its role in development remains obscure. To understand its function during plant development, we studied the effects of loss and gain of function. Expression of ATHB12 fused to the EAR-motif repression domain (SRDX) - P35 S ::ATHB12SRDX (A12SRDX) and PATHB 12 ::ATHB12SRDX - slowed both leaf and root growth, while the growth of ATHB12-overexpressing seedlings (A12OX) was accelerated. Microscopic examination revealed changes in the size and number of leaf cells. Ploidy was reduced in A12SRDX plants, accompanied by decreased cell expansion and increased cell numbers. By contrast, cell size was increased in A12OX plants, along with increased ploidy and elevated expression of cell cycle switch 52s (CCS52s), which are positive regulators of endoreduplication, indicating that ATHB12 promotes leaf cell expansion and endoreduplication. Overexpression of ATHB12 led to decreased phosphorylation of Arabidopsis thaliana ribosomal protein S6 (AtRPS6), a regulator of cell growth. In addition, induction of ATHB12 in the presence of cycloheximide increased the expression of several genes related to cell expansion, such as EXPANSIN A10 (EXPA10) and DWARF4 (DWF4). Our findings strongly suggest that ATHB12 acts as a positive regulator of endoreduplication and cell growth during leaf development.

Long-term comparative study of Substance-P with methylprednisolone on the development of osteoporosis.

Steroids are treated for most inflammatory diseases but cause serious side effects such as diabetes and osteoporosis after their long-term usage. Recently, we identified novel roles of Substance-P (SP) in the suppression of the injury-mediated inflammation and also in stem cell mobilization. In this study, for clinical application of SP as an anti-inflammatory agent, its safety in long-term usage was evaluated with regard to diabetes and osteoporosis. Dexamethasone (DEX) and methylprednisolone (MP) were used as comparative drugs. While DEX-injection for 24 weeks developed severe weight loss, unstable blood glucose, and bone loss, SP-injection did not affect blood glucose and bone mass. MP-injection for 24 weeks also influenced blood glucose and body weight much milder than DEX-injection. After 66 weeks, MP-injection caused unstable blood glucose, alleviation in the age-related increase of body weight, and bone weakness, which was featured by reduction in collagen deposition and trabecular bone volume based on histological and micro CT analysis. However, SP-injection for 66 weeks rather increased collagen deposition, bone volume, and bone density. Therefore, this comparative study suggests that SP, even after long-term usage of effective dose, may not cause side effects such as osteoporosis in comparison to that of DEX and MP and can be developed as an anti-inflammatory agent and/or stem cell mobilizer for long-term treatment.

Functional implication of ß-carotene hydroxylases in soybean nodulation.

Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative ß-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

RNA interference-mediated repression of S6 kinase 1 impairs root nodule development in soybean.

Symbiotic nodule formation on legume roots is characterized with a series of developmental reprograming in root tissues, including extensive proliferation of cortical cells. We examined a possible involvement of the target of rapamycin (TOR) pathway, a central regulator of cell growth and proliferation in animals and yeasts, during soybean nodule development. Our results show that transcription of both GmTOR and its key downstream effector, GmS6K1, are activated during nodulation, which is paralleled with higher kinase activities of these gene products as well. RNAi-mediated knockdown of GmS6K1 impaired the nodule development with severely reduced nodule weight and numbers. In addition, expression of a few nodulins including leghemoglobin was also decreased, and consequently nitrogen fixation was found to be reduced by half. Proteomic analysis of the GmS6K1-RNAi nodules identified glutamine synthetase (GS), an essential enzyme for nitrogen assimilation in nodules, as one of the proteins that are significantly down regulated. These results appear to provide solid evidence for a functional link between GmS6K1 and nodule development.