5-FU-miR-15a Inhibits Activation of Pancreatic Stellate Cells by Reducing YAP1 and BCL-2 Levels In Vitro.

Chronic pancreatitis is characterized by chronic inflammation and fibrosis, processes heightened by activated pancreatic stellate cells (PSCs). Recent publications have demonstrated that miR-15a, which targets YAP1 and BCL-2, is significantly downregulated in patients with chronic pancreatitis compared to healthy controls. We have utilized a miRNA modification strategy to enhance the therapeutic efficacy of miR-15a by replacing uracil with 5-fluorouracil (5-FU). We demonstrated increased levels of YAP1 and BCL-2 (both targets of miR-15a) in pancreatic tissues obtained from Ptf1aCreERTM and Ptf1aCreERTM;LSL-KrasG12D mice after chronic pancreatitis induction as compared to controls. In vitro studies showed that delivery of 5-FU-miR-15a significantly decreased viability, proliferation, and migration of PSCs over six days compared to 5-FU, TGFß1, control miR, and miR-15a. In addition, treatment of PSCs with 5-FU-miR-15a in the context of TGFß1 treatment exerted a more substantial effect than TGFß1 alone or when combined with other miRs. Conditioned medium obtained from PSC cells treated with 5-FU-miR-15a significantly inhibits the invasion of pancreatic cancer cells compared to controls. Importantly, we demonstrated that treatment with 5-FU-miR-15a reduced the levels of YAP1 and BCL-2 observed in PSCs. Our results strongly suggest that ectopic delivery of miR mimetics is a promising therapeutic approach for pancreatic fibrosis and that 5-FU-miR-15a shows specific promise.

AMPK-mediated activation of Akt protects renal tubular cells from stress-induced apoptosis in vitro and ameliorates ischemic AKI in vivo.

We have reported that preconditioning renal tubular cells (RTCs) with A-769662 [a pharmacological activator of AMP-activated protein kinase (AMPK)] reduces apoptosis of RTCs induced by subsequent stress and ameliorates the severity of ischemic acute kidney injury (AKI) in mice. In the present study, we examined the role of the phosphoinositide 3-kinase (PI3K)/Akt pathway in mediating these effects. Using shRNA, we developed knockdown (KD) RTCs to confirm that any novel effects of A-769662 are mediated specifically by AMPK. We reduced expression of the total ß-domain of AMPK in KD RTCs by >80%. Control RTCs were transfected with "scrambled" shRNA. Preconditioning control RTCs with A-769662 increased both the phosphorylation (activity) of AMPK and survival of these cells when exposed to subsequent stress, but neither effect was observed in KD cells. These data demonstrate that activation of AMPK by A-769662 is profoundly impaired in KD cells. A-769662 activated PI3K and Akt in control but not KD RTCs. These data provide novel evidence that activation of the PI3K/Akt pathway by A-769662 is mediated specifically through activation of AMPK and not by a nonspecific mechanism. We also demonstrate that, in control RTCs, Akt plays a role in mediating the antiapoptotic effects of A-769662. In addition, we provide evidence that AMPK ameliorates the severity of ischemic AKI in mice and that this effect is also partially mediated by Akt. Finally, we provide evidence that AMPK activates PI3K by inhibiting mechanistic target of rapamycin complex 1 and preventing mechanistic target of rapamycin complex 1-mediated inhibition of insulin receptor substrate-1-associated activation of PI3K.

Apoptotic cells activate AMP-activated protein kinase (AMPK) and inhibit epithelial cell growth without change in intracellular energy stores.

Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses.

Preconditioning mice with activators of AMPK ameliorates ischemic acute kidney injury in vivo.

This study had two objectives: 1) to determine whether preconditioning cultured proximal tubular cells (PTCs) with pharmacological activators of AMP-activated protein kinase (AMPK) protects these cells from apoptosis induced by metabolic stress in vitro and 2) to assess the effects of preconditioning mice with these agents on the severity of ischemic acute renal kidney injury (AKI) in vivo. We demonstrate that preconditioning PTCs with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) or A-769662 reduces apoptosis of PTCs induced by subsequent stress. We also show that the reduction in cell death during metabolic stress associated with pretreatment by AMPK activators is associated with an increase in the cytosolic level of ATP, which is mediated by an increase in the rate of glycolysis. In addition, we provide evidence that the effect of AMPK activators on glycolysis is mediated, at least in part, by an increased uptake of glucose, and by the induction of hexokinase II (HK II) expression. Our data also show that the increased in HK II expression associated with preconditioning with AMPK activators is mediated by the activation (phosphorylation) of the cAMP-response element binding protein (CREB). We also provide entirely novel evidence that that A-79662 is substantially more effective than AICAR in mediating these alterations in PTCs in vitro. Finally, we demonstrate that preconditioning mice with AICAR or A-769662 substantially reduces the severity of renal dysfunction and tubular injury in a model of ischemic AKI in vivo and that the efficacy of AICAR and A-768662 in ameliorating ischemic AKI in vivo is comparable.

Collective total synthesis of stereoisomeric yohimbine alkaloids.

Stereoisomeric polycyclic natural products are important for drug discovery-based screening campaigns, due to the close correlation of stereochemistry with diversified bioactivities. Nature generates the stereoisomeric yohimbine alkaloids using bioavailable monoterpene secolaganin as the ten-carbon building block. In this work, we reset the stage by the development of a bioinspired coupling, in which the rapid construction of the entire pentacyclic skeleton and the complete control of all five stereogenic centers are achieved through enantioselective kinetic resolution of an achiral, easily accessible synthetic surrogate. The stereochemical diversification from a common intermediate allows for the divergent and collective synthesis of all four stereoisomeric subfamilies of yohimbine alkaloids through orchestrated tackling of thermodynamic and kinetic preference.

Susceptibility to ATP depletion of primary proximal tubular cell cultures derived from mice lacking either the α1 or the α2 isoform of the catalytic domain of AMPK.

BACKGROUND: The purpose of this study was to determine whether AMPK influences the survival of primary cultures of mouse proximal tubular (MPT) cells subjected to metabolic stress. Previous studies, using an immortalized MPT cell line, suggest that AMPK is activated during metabolic stress, and ameliorates stress-induced apoptosis of these cells. METHODS: Primary MPT cells were cultured from AMPK knockout (KO) mice lacking either the α1 or the α2 isoform of the catalytic domain of AMPK. MPT cells were subjected to ATP depletion using antimycin A. RESULTS: Surprisingly, there was no difference in the amount of death induced by metabolic stress of MPT cells from either type of AMPK KO mice compared to its WT control. Moreover, inhibition of the activity of the α1 isoform in primary MPT cells from α2-/- mice (pharmacologically, via compound C) or inhibition of the α2 isoform in primary MPT cells from α1-/- mice (molecularly, via knockdown) both decreased cell viability equivalently in response to metabolic stress. The explanation for this unexpected result appears to be an adaptive increase in expression of the non-deleted α-isoform. As a consequence, total α-domain expression (i.e. α1 + α2), is comparable in kidney cortex and in cultured MPT cells derived from either type of KO mouse versus its WT control. Importantly, each α-isoform appears able to compensate fully for the absence of the other, with respect to both the phosphorylation of downstream targets of AMPK and the amelioration of stress-induced cell death. CONCLUSIONS: These findings not only confirm the importance of AMPK as a pro-survival kinase in MPT cells during metabolic stress, but also show, for the first time, that each of the two α-isoforms can substitute for the other in MPT cells from AMPK KO mice with regard to amelioration of stress-induced loss of cell viability.

Krüppel-like Factor 5 Plays an Important Role in the Pathogenesis of Chronic Pancreatitis.

Chronic pancreatitis results in the formation of pancreatic intraepithelial neoplasia (PanIN) and poses a risk of developing pancreatic cancer. Our previous study demonstrated that Krüppel-like factor 5 (KLF5) is necessary for forming acinar-to-ductal metaplasia (ADM) in acute pancreatitis. Here, we investigated the role of KLF5 in response to chronic injury in the pancreas. Human tissues originating from chronic pancreatitis patients showed increased levels of epithelial KLF5. An inducible genetic model combining the deletion of Klf5 and the activation of KrasG12D mutant expression in pancreatic acinar cells together with chemically induced chronic pancreatitis was used. The chronic injury resulted in increased levels of KLF5 in both control and KrasG12D mutant mice. Furthermore, it led to numerous ADM and PanIN lesions and extensive fibrosis in the KRAS mutant mice. In contrast, pancreata with Klf5 loss (with or without KrasG12D) failed to develop ADM, PanIN, or significant fibrosis. Furthermore, the deletion of Klf5 reduced the expression level of cytokines and fibrotic components such as Il1b, Il6, Tnf, Tgfb1, Timp1, and Mmp9. Notably, using ChIP-PCR, we showed that KLF5 binds directly to the promoters of Il1b, Il6, and Tgfb1 genes. In summary, the inactivation of Klf5 inhibits ADM and PanIN formation and the development of pancreatic fibrosis.

The aggregation behaviour and mechanism of commercial graphene oxide in surface aquatic environments.

In this study, we comprehensively and critically discuss the aggregation mechanism of commercial graphene oxide (CGO) in surface aquatic environments. The aggregation kinetics and critical coagulation concentration of CGO were obtained through time-resolved dynamic light scattering and batch techniques over a wide range of water types. By employing transmission electron microscopy and elemental mapping, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy, we studied the effects of cations in natural waters on the microstructure transformation, element content and distribution, and oxygen-containing functional group vibrations of CGO. The aggregation of CGO in natural water is induced mainly by Ca2+ by complexing; Na+, with a higher concentration, plays a more important role than Mg2+ in inducing aggregation via electric double layer suppression. Ca2+ mainly interacts with C - COOH, while Mg2+ has a greater effect on C - OH. Na+ has less effect on the oxygen-containing functional group but decreases the C/O ratio in contrast with Mg2+/Ca2+/natural water, indicating the different inducing mechanisms. This study looks forward to providing pivotal knowledge to predict the environmental fate of CGO more accurately in natural surface water.

Efficient heavy metal removal from water by alginate-based porous nanocomposite hydrogels: The enhanced removal mechanism and influencing factor insight.

Novel porous alginate-based nanocomposite hydrogels were prepared by incorporating polyaniline-polypyrrole modified graphene oxide (GO@PAN-PPy) as reinforcing fillers into the alginate matrix (GO@PAN-PPy/SA) for Cr(VI) and Cu(II) removal from water. Different in-situ co-polymerization functionalized GO with Py-to-An mass ratios of monomers (from nil to 1:1) and contents of GO@PAN-PPy (from nil to 2.0%(w/v)) were embedded into the alginate backbone to improve the sorption performance. Key factors, such as pH, coexisting metal ions, and swelling states were investigated in batch adsorption modes. The synergistic effect combined from polymer backbone and fillers could lower the impact of the pH-dependent adsorption reaction. With an adsorption ability superior to that of plain SA and GO/SA, the optimized GO@PAN-PPy-2(1)/SA exhibited good experimental maximum adsorption capacities for Cr(VI) (~133.7 mg/g) and Cu(II) (~87.2 mg/g) at pH 3.0, which were better than those of many other similar sorbents. The sorbents possessed excellent adaptability for 0.2 M salt for Cr(VI) removal but poor for Cu(II) removal. Pre-swelling treatment and co-adsorption could enhance the adsorption performance. The excellent reusability of hydrogel was demonstrated after five cycles in single/binary system. Overall, this work reveals that the resultant hydrogel holds potential as candidate sorbent to remove anionic-cationic heavy metal ions from water.

Insights into the simultaneous nitrification, denitrification and phosphorus removal process for in situ sludge reduction and potential phosphorus recovery.

A simultaneous nitrification-denitrification and phosphorus removal (SNDPR) system operated in an alternating anaerobic/aerobic/anoxic (A/O/A) mode was revisited from new perspectives of sludge reduction and potential phosphorus recovery. Reliable and robust removal performance was obtained even under winter temperatures, with average removal efficiency of COD, TP, NH4+-N and TIN being 89.68%, 93.60%, 92.15% and 79.01% at steady state, respectively. Inoculated sludge got enhanced in biomass density, settleability, and bioactivity. And relatively stable amounts of extracellular polymeric substances (EPS) with a stable protein/ polysaccharide (PN/PS) ratio were observed over operation. Meanwhile, a low observed sludge yield (Yobs) of 0.083 g MLSS/g COD (0.082 g MLVSS/g COD) was obtained. A maximum anaerobic phosphorus release up to 43.54 mg/L was found, thus providing phosphorus-rich and low-turbidity stream for further phosphorus recovery. Overall, the SNDPR system deserved attention for in situ sludge reduction and potential phosphorus recovery, beyond reliable and stable wastewater treatment.

Lefty peptides, derived by MMP2 cleavage, act as a new class of gelatinase A inhibitor.

MMPs are zinc-dependent endopeptidases that are involved in proteolysis of extracellular matrix in both physiological and pathological processes including cancer. MMPs are involved at all stages of tumor progression, including tumor growth, angiogenesis, and metastasis. We recently showed that overexpression of Lefty in cancer cells restrains tumor growth. Here, we show that small forms of Lefty are generated by MMP2 (gelatinase A) mediated cleavage. In turn, these forms of Lefty strongly inhibit the autocatalytic, gelatinolytic and caseinolytic activities of MMP2 in vitro. We show that a short synthesized form of Lefty peptide (CASDGALVP) inhibits gelatinolytic and caseinolytic activities of MMP2 in vitro and inhibits tumor growth in vivo. Together, these findings show that lefty peptides are a new class of gelatinase A inhibitors that restrain tumor growth.

Tet responsive and adenovirus based constructs for regulated in vivo expression of Lefty.

Regulated expression of Lefty/Ebaf during embryogenesis is required for development of body asymmetry. A tight regulation of Lefty also contributes to the menstrual tissue shedding in humans. In order to replicate this regulated expression, we have developed a tet-on system and an adenovirus driven model. To drive the expression of Lefty, we have placed Lefty under control of a tetracycline-responsive promoter which responds to a sequence variant of the reversed tet transcriptional activator (rtTA), rtTA2S-M2. In this model, Lefty is regulated by the dose of doxycycline. In the adenovirus driven system, Lefty is regulated by the number of adenoviral particles These model systems would be suitable for understanding the dose-dependent biologic role of Lefty in vivo.

Decidual differentiation of stromal cells promotes Proprotein Convertase 5/6 expression and lefty processing.

Lefty/Ebaf polypeptides, novel members of the TGF-beta superfamily, are involved in endometrial differentiation and embryo implantation. Recently, we showed that, during undisturbed estrous cycle, lefty is present in mouse uterine horn primarily in a precursor form. Here, we show that decidual differentiation of endometrial stroma leads to increased lefty (approximately 3.1- to 3.6-fold in vivo and 5- to 8-fold in vitro) and processing of its precursor primarily to its long form. This event occurs on d 5 of pregnancy, and is paralleled by proprotein convertase (PC)5/6 up-regulation (approximately 6-fold increase for PC5A and 3-fold increase for PC5B) in decidualized uterine horn, independent of embryo implantation. Among the known convertases, only PC5/6A processes lefty to its long form. Taken together, the findings show that decidualized differentiation of stroma, which is a prerequisite for embryo implantation, leads to processing of lefty by PC5/6A.

Modeling gene expression in the mouse uterine horn by in vivo adenovirus-mediated gene delivery.

Endometrium is a unique tissue that is prepared for implantation of blastocyst during each menstrual cycle by expression of genes during a defined period of endometrial receptivity. Induction of gene over-expression in endometrium allows gaining insight on the role that genes play in endometrial function. Here, we show that induction of a state of gene over-expression in endometrium is feasible by in vivo gene delivery by transduction with adenovirus. Analysis of endometrium following adenoviral transduction of LacZ showed increased beta-galactosidase activity in endometrial glands as early as 24 hours following in vivo gene transfer. By 72 hour, the expression was uniformly strong throughout the uterine horn. Viral transduction was efficient in the range of 0.24-24x10(8) pfu (4.8-480x10(8) particles) in normal, pregnant and decidualized mouse uterine horns. These findings show that induction of a state of gene over-expression can be successfully attained in endometrium by adenoviral gene delivery.

Progesterone receptor activates its promoter activity in human endometrial stromal cells.

Previous studies have shown that progestin increases the content of progesterone receptor (hPRA and hPRB) and the hPR mRNA during decidualization of human endometrial stromal cells suggesting that endogenous hPR enhances the transcription of the hPR gene. In the present study, we provide evidence that hPR regulates the promoter activity mediated through an active Sp1 site. In stromal cells treated with medroxyprogesterone acetate, the promoter activity was significantly increased when cells were co-transfected with hPR expression vector. Progressive deletion analysis showed that the highest activity was derived from the promoter region between -55 and +31 bp. Transactivation by hPR was dose dependent. The capacity of hPRA was stronger than that of hPRB. The ligand binding domain, but not DNA binding domain of the hPR was required for the transactivation. The proximal promoter region lacks a canonical progesterone response element. Instead, an active Sp1 site (-49 to -43 bp) has been confirmed. Mutation of the Sp1 site eliminated the effect of hPR activation. The promoter activity was increased by over expression of Sp1, whereas Sp3 had no effect. Electrophoretic mobility shift assay showed that the promoter region between -55 and +31 bp bound to Sp1 family proteins, Sp1 (C2 complex) and Sp3 (C1 and C3 complexes) identified by antibodies to Sp1 and Sp3. Sp1 complex formed by extracts of stromal cells was less intense than that formed by progestin-decidualized stromal cells. Sp1/DNA binding was enhanced when stromal cell extracts were incubated with calf intestine alkaline phosphatase (CIP) suggesting that dephosphorylation of Sp1 enhances the DNA binding. Addition of protein kinase inhibitor, H-89 or H-7, enhanced the hPR stimulated promoter activity. Western blot analysis showed that endometrial stromal/decidual cell extracts contained a wide band of Sp1 spanning from approximately 105 to 96 kDa and was resolved into one band at 96 kDa by CIP. Decidual cell extracts are abundant with the 96 kDa Sp1. In addition, the 96 kDa Sp1 was co-precipitated with ligand-activated hPRA or hPRB in the decidual cell nuclear extracts. These data suggest that dephosphorylated Sp1, abundant in decidual cells, enhances the binding to both DNA and hPR resulting in a robust increase of the hPR promoter activity.

Progesterone receptor (hPR) upregulates the fibronectin promoter activity in human decidual fibroblasts.

Previous studies have shown that progestin induces the production of fibronectin (FN) and its mRNA content in human endometrial stromal cells. The mechanism of the upregulation was unclear. In the present study, we provide evidence that hPR regulates the FN promoter activity mainly through the CRE/AP1 site located in the proximal region of the promoter in human decidual fibroblasts. Various lengths of the proximal region of the FN promoter were linked to the reporter vector to construct promoter-reporter plasmids and were then transfected into human decidual fibroblasts. Deletion and mutation analysis showed that CRE/AP1 and Sp1 sites in the proximal region mediated the basal promoter activity. To evaluate progestin-mediated transcriptional activation, decidual fibroblasts were transfected with p300 (FN promoter-reporter construct) and hPR expression vector. Cells treated with medroxyprogesterone acetate (MPA) increased the promoter activity ranging from 2.5- to 9-fold determined in 10 decidual specimens. hPRA enhanced activation was stronger than that of hPRB. Structural analysis of hPR showed that DNA and ligand binding domains are essential for the activation, and missing the TAF1 domain weakens the activation. The proximal promoter region of the FN gene lacks a canonical PRE site. Mutation at the CRE/AP1 site eliminated the upregulation by progestin. To evaluate the interaction of hPR with the CRE/AP1 site, the CRE/AP1 site was mutated to the consensus AP1 cis-element (TGACGTCA, -172 to -165 bp, mutated to TGAC_TCA) which eliminated the CREB binding. FN promoter activity derived from p300AP1 mutant was found to be higher than that of p300. These results showed that hPR interacts with the AP1 binding proteins, but not with CREB. Progestin treatment or overexpression hPR did not alter appreciably the content of c-jun or c-fos in decidual fibroblasts nuclear extracts. Antibody to hPR (hPRa3), which precipitated hPR also coprecipitated c-jun and c-fos, whereas CREB was not precipitated by hPRa3. The observation implies that hPRs are brought to the FN promoter region by AP1 proteins to enhance the transcription. In summary, this study provides molecular evidence that the CRE/AP1 site and c-jun/c-fos in decidual fibroblasts mediate the hPR-enhanced activation of FN transcription.

LEFTY, a member of the transforming growth factor-beta superfamily, inhibits uterine stromal cell differentiation: a novel autocrine role.

LEFTY is expressed in normal endometrium in cells that decidualize. To understand the importance of this expression, we have studied the effect of LEFTY on decidualization in vitro and in vivo. Exposure of human uterine fibroblast (HuF) cells to recombinant LEFTY blocked the induction of the decidual differentiation-specific marker genes, IGFBP1 (IGF-binding protein 1) and PRL (prolactin) in response to medroxyprogesterone acetate, estradiol, and prostaglandin E2. The inhibitory effect was associated with decreased induction of the transcription factors ETS1 and FOXO1, both of which are essential for decidualization. Overexpression of LEFTY in decidualized HuF cells with an adenovirus that transduced LEFTY caused a marked decrease in IGFBP1 secretion, and withdrawal of medroxyprogesterone acetate from decidualized cells resulted in a decrease in IGFBP1 secretion and an increase in LEFTY expression. Moreover, overexpression of LEFTY in decidualized cells reprogrammed the cells to a less differentiated state and attenuated expression of decidual markers. Uterine decidualization was markedly attenuated and litter size was significantly reduced by retroviral transduction of LEFTY in the uterine horns of pregnant mice or by induction of LEFTY expression by doxycycline treatment in Tet-On conditional LEFTY transgenic pregnant mice. In addition, administration of the contraceptive agent drospirenone to ovariectomized mice induced a marked increase in LEFTY expression and inhibited decidualization. Taken together, these finding indicate that LEFTY acts as a molecular switch that modulates both the induction of decidual differentiation and the maintenance of a decidualized state. Because decidual cells express abundant amounts of LEFTY, the action of LEFTY on decidualization occurs by an autocrine mechanism.

The regulated cell surface zymogen activation of the proprotein convertase PC5A directs the processing of its secretory substrates.

The proprotein convertases are synthesized as zymogens that acquire activity upon autocatalytic removal of their NH(2)-terminal prosegment. Based on the convertase furin, to fold properly and gain activity, the convertases PC5A, PACE4, and PC7 are presumed to undergo two sequential prosegment cleavages in the endoplasmic reticulum and then in the trans-Golgi network. However, biochemical and immunocytochemical experiments revealed that mouse PC5A is complexed to its prosegment at the plasma membrane. This labeling is lost upon treatment with heparin and is increased by overexpressing members of the syndecan family and CD44, suggesting attachment of secreted PC5A-prosegment complex to heparan sulfate proteoglycans. Following stimulation of Y1 cells with adrenocorticotropic hormone or 8-bromo-cyclic AMP, the cell surface labeling of the prosegment of PC5A is greatly diminished, whereas the signal for mature PC5A is increased. Moreover, after stimulation, the protease activity of PC5A is enhanced, as evidenced by the cleavage of the PC5A substrates Lefty, ADAMTS-4, endothelial lipase, and PCSK9. Our data suggest a novel mechanism for PC5A activation and substrate cleavage at the cell surface, through a regulated removal of its prosegment. A similar mechanism may also apply to the convertase PACE4, thereby extending our knowledge of the molecular details of the zymogen activation and functions of these heparan sulfate proteoglycan-bound convertases.

In vivo gene transfer of lefty leads to implantation failure in mice.

BACKGROUND: Endometrium is a unique tissue that is prepared for implantation of blastocyst during each menstrual cycle. In humans, if implantation does not occur or fails, endometrium is shed. METHODS AND RESULTS: We identified ebaf/lefty, as a key cytokine, highly expressed in human endometrium during the non-receptive phase of tissue remodelling. Lefty was increased in the endometria of a number of patients with 'unexplained infertility' during the receptive phase, suggesting dysregulation of lefty as a potential factor contributing to infertility. Here, we showed that induction of a similar state of lefty overexpression in endometrium, by in vivo gene delivery, decreased implantation in pregnant mice. This state of overexpression could be induced by a retroviral vector transducing lefty or by liposome-mediated introduction of a lefty expression vector. Analysis of endometrium showed increased lefty after in vivo gene transfer. CONCLUSION: These findings suggest that induction of a state of lefty overexpression in endometrium leads to reduced implantation.

Ligand activated relaxin receptor increases the transcription of IGFBP-1 and prolactin in human decidual and endometrial stromal cells.

The aim of this study was to investigate relaxin (RLX) receptor-mediated gene activation in human endometrium. We determined the promoter activities of insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin (PRL) and identified sequence(s) that mediate RLX activated transcription in human decidual cells and endometrial stromal cells. In human decidual cells, the promoter activity of IGFBP-1 was increased significantly in cells incubated with RLX. In endometrial stromal cells, the RLX mediated activation was enhanced only when stromal cells were co-transfected with RLX-receptor (LGR7) expression vector and RLX alone had little effect (Mazella et al., 2004). Deletion and mutation analysis showed that the cAMP regulatory element (CRE, -263 to -259 bp) in the IGFBP-1 promoter was essential for the activation. In addition, RLX increased the phosphorylation of CRE binding protein (CREB to p-CREB) and p-CREB resided in the nucleus, indicating that RLX activates the protein kinase (PKA) system in decidual cells. Gel shift assay showed that nuclear extracts prepared from RLX treated decidual cells increased the binding to the CRE site of the IGFBP-1 promoter. RLX increased the PRL promoter activity mediated through the region containing multiple CCAAT/enhancer-binding proteins (C/EBP) binding sites that have been shown to mediate the PRL gene activation by cAMP analogue (Pohnke et al., 1999). RLX enhanced IGFBP-1 promoter activity was inhibited by cAMP dependent PKA inhibitor, H-89. PRL promoter activity was inhibited by both H-89 and U0126 indicating multiple signalling pathways are activated by RLX in endometrial cells for different target gene activation.