Engineering a single-chain vascular endothelial growth factor with enhanced physiological stability for accelerating wound healing.

Vascular endothelial growth factor (VEGF) is characterized by its homodimeric structure, in which two cysteine-knot subunits are covalently linked. Although its profound pro-angiogenic activity presents considerable therapeutic promise, challenges associated with dimer stability and its short physiological half-life restrict its clinical translation. To address these limitations, we engineered prototype VEGF-A165 into a single-chain construct, V165-CTP-V165, by incorporating the C-terminal peptide (CTP) of human chorionic gonadotropin as a linker to enhance dimerization efficiency and improve protein stability of VEGF.

Unveiling the signal valve specifically tuning the TGF-β1 suppression of osteogenesis: mediation through a SMAD1-SMAD2 complex.

Background: TGF-β1 is the most abundant cytokine in bone, in which it serves as a vital factor to interdict adipogenesis and osteogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, how TGF-β1 concurrently manipulates differentiation into these two distinct lineages remains elusive.

Methods: Treatments with ligands or inhibitors followed by biochemical characterization, reporter assay, quantitative PCR and induced differentiation were applied to MSC line or primary BM-MSCs for signaling dissection.

Unveiling the transcriptomic landscape and the potential antagonist feedback mechanisms of TGF-β superfamily signaling module in bone and osteoporosis.

Background: TGF-β superfamily signaling is indispensable for bone homeostasis. However, the global expression profiles of all the genes that make up this signaling module in bone and bone-related diseases have not yet been well characterized.

Methods: Transcriptomic datasets from human bone marrows, bone marrow-derived mesenchymal stem cells (MSCs) and MSCs of primary osteoporotic patients were used for expression profile analyses.

Expression profiling of ovarian BMP antagonists reveals the potential interaction between TWSG1 and the chordin subfamily in the ovary.

The TGF-β superfamily members and their antagonists comprise an indispensable system that controls mammalian ovarian development in a sophisticated manner. In contrast to a plethora of studies on the ovary-expressed TGF-β superfamily members, knowledge regarding their antagonists, including their expression profiles and antagonism preferences, is still lacking. Using quantitative PCR in rats and transcriptomic dataset comparisons in mice and humans, we set out to characterize the relative expression levels of most antagonists in the mammalian ovary.

Human bone morphogenetic protein 8A promotes expansion and prevents apoptosis of cumulus cells in vitro.

Cumulus expansion is essential for ovulation and oocyte maturation in mammals. Previous studies suggest that this process requires certain cumulus expansion enabling factors, induced by LH surge, that activate SMAD signaling locally. However, their identities remain uncertain.

Human BMP8A suppresses luteinization of rat granulosa cells via the SMAD1/5/8 pathway.

Bone morphogenetic proteins (BMPs) are known to play an indispensable role in preventing the precocious luteinization of granulosa cells within growing ovarian follicles. In this study, we found that the transcripts of BMP8 genes are enriched in the ovaries of humans and rodents. When analyzing transcriptomic datasets obtained from human mature granulosa cells, we further found that the BMP8 transcripts not only show the highest abundance among the searchable BMP-related ligands but also decrease significantly in women of advanced age or women with polycystic ovarian syndrome.

In vivo selection reveals autophagy promotes adaptation of metastatic ovarian cancer cells to abdominal microenvironment.

Peritoneal dissemination is the most frequent metastatic route of ovarian cancer. However, due to the high heterogeneity in ovarian cancer, most conventional studies lack parental tumor controls relevant to metastases and, thus, it is difficult to trace the molecular changes of cancer cells along with the selection by the abdominal microenvironment. Here, we established an in vivo mouse peritoneal dissemination scheme that allowed us to select more aggressive sublines from parental ovarian cancer cells, including A2780 and SKOV-3.

BMP8A sustains spermatogenesis by activating both SMAD1/5/8 and SMAD2/3 in spermatogonia.

Mutation in either of the genes encoding bone morphogenetic protein (BMP) 8A or 8B ( or ) causes postnatal depletion of spermatogonia in mice. We found that , but not , was expressed predominantly in the neonatal mouse spermatogonia. Although most BMPs induce activation of SMADs 1, 5, and 8 (SMAD1/5/8), but not SMADs 2 and 3 (SMAD2/3), we found that BMP8A induced signaling through both sets of transcription factors.

Thyrostimulin-TSHR signaling promotes the proliferation of NIH:OVCAR-3 ovarian cancer cells via trans-regulation of the EGFR pathway.

Gonadotropin signaling plays an indispensable role in ovarian cancer progression. We previously have demonstrated that thyrostimulin and thyroid-stimulating hormone receptor (TSHR), the most ancient glycoprotein hormone and receptor pair that evolved much earlier than the gonadotropin systems, co-exist in the ovary. However, whether thyrostimulin-driven TSHR activation contributes to ovarian cancer progression in a similar way to gonadotropin receptors has never been explored.

NMU signaling promotes endometrial cancer cell progression by modulating adhesion signaling.

Neuromedin U (NMU) was originally named based on its strong uterine contractile activity, but little is known regarding its signaling/functions in utero. We identified that NMU and one of its receptors, NMUR2, are not only present in normal uterine endometrium but also co-expressed in endometrial cancer tissues, where the NMU level is correlated with the malignant grades and survival of patients. Cell-based assays further confirmed that NMU signaling can promote cell motility and proliferation of endometrial cancer cells derived from grade II tumors.

Identifying a Neuromedin U Receptor 2 Splice Variant and Determining Its Roles in the Regulation of Signaling and Tumorigenesis In Vitro.

Neuromedin U (NMU) activates two G protein-coupled receptors, NMUR1 and NMUR2; this signaling not only controls many physiological responses but also promotes tumorigenesis in diverse tissues. We recently identified a novel truncated NMUR2 derived by alternative splicing, namely NMUR2S, from human ovarian cancer cDNA. Sequence analysis, cell surface ELISA and immunocytochemical staining using 293T cells indicated that NMUR2S can be expressed well on the cell surface as a six-transmembrane protein.

A naturally occurring Lgr4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of Lgr4 in mammals.

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals.

Multi-functional norrin is a ligand for the LGR4 receptor.

Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs.

Ovarian regulation of neuromedin U and its local actions in the ovary, mediated through neuromedin U receptor 2.

Neuromedin U (NMU) was originally identified as an anorexigenic peptide that modulates appetite as well as energy homeostasis through the brain-gut axis. Although growing evidence has linked NMU activity with the development of female reproductive organs, no direct expression of and function for NMU in these organs has been pinpointed. Using a superovulated rat model, we found that NMU is directly expressed in the ovary, where its transcript level is tightly regulated by gonadotropins.

CXCL17, an orphan chemokine, acts as a novel angiogenic and anti-inflammatory factor.

Chemokines play pivotal roles in the recruitment of various immune cells to diverse tissues in both physiological and pathological conditions. CXCL17 is an orphan chemokine preliminarily found to be involved in tumor angiogenesis. However, its protein nature, as well as its endogenous bioactivity, has not been well clarified.

DAN (NBL1) specifically antagonizes BMP2 and BMP4 and modulates the actions of GDF9, BMP2, and BMP4 in the rat ovary.

Although differential screening-selected gene aberrative in neuroblastoma (DAN, official symbol NBL1) is the founding member of the DAN subfamily of bone morphogenetic protein (BMP) antagonists, its antagonizing targets, gene regulation, and physiological functions remain unclear. Using diverse cell expression systems, we found that the generation of bioactive DAN is likely to be cell type specific. Unlike other phylogenetically close members, which are covalently linked homodimers, DAN forms a noncovalently linked homodimer during folding.

Mutual adaptation between mouse transglutaminase 4 and its native substrates in the formation of copulatory plug.

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I-III, which are the major components of mouse SVS.

Exclusive expression of a membrane-bound Spink3-interacting serine protease-like protein TESPL in mouse testis.

We identified a testis-specific protease-like protein tentatively named TESPL and a pancreatic trypsinogen Prss2 from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. The enzymatic motifs and the cysteine patterns in serine proteases are highly conserved in these two proteins. Based on the phylogenetic analysis, Prss2 duplicated recently and TESPL underwent distant evolution without gene duplication from the progenitor of trypsin-like and chymotrypsin-like proteases.

Thyrostimulin, but not thyroid-stimulating hormone (TSH), acts as a paracrine regulator to activate the TSH receptor in mammalian ovary.

The thyroid-stimulating hormone receptor (TSHR), activated by either TSH or the newly discovered glycoprotein hormone thyrostimulin, plays a central role in the control of body metabolism. Interestingly, in addition to its thyroid expression, we discovered that the mRNA level of TSHR is periodically regulated in rat ovary by gonadotropins. Ovarian microdissection followed by real-time PCR analysis indicated that granulosa cells show the highest level of TSHR expression.

Establishment of a chimeric reporting system for the universal detection and high-throughput screening of G protein-coupled receptors.

G proteins, further divided into four subfamilies (G(s), G(q), G(12) and G(i)) based on their Galpha subunits, are the primary components activated by G protein-coupled receptors (GPCRs). Current GPCR assays are limited to the evaluation of selective Galpha signaling and do not allow comprehensive screening for orphan GPCRs without a known coupled Galpha. Therefore, our aim was to design a chimeric reporting system that covers responses from all Galpha subfamilies simultaneously.

Crystallization and preliminary X-ray diffraction analysis of PAT, an acetyltransferase from Sulfolobus solfataricus.

PAT is an acetyltransferase from the archaeon Sulfolobus solfataricus that specifically acetylates the chromatin protein Alba. The enzyme was expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data were collected to 1.

Molecular dissection of G protein preference using Gsalpha chimeras reveals novel ligand signaling of GPCRs.

Although only 16 genes have been identified in mammals, several Galpha subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Galpha activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G(s)alpha and the carboxyl termini of the various Galpha proteins determine their receptor coupling specificity, we proposed a set of chimeric G(s)alpha where the COOH-terminal five amino acids are replaced by those of other Galpha proteins and used these to dissect the potential Galpha linked to a given GPCR.

Epitope topology and removal of mouse acrosomal plasma membrane by P12-targeted immunoaggregation.

P12 is a Kazal-type trypsin inhibitor that has been purified from mouse seminal vesicle secretion. We observed a slight impact of P12 on sperm capacitation, and demonstrated the removal of plasma membrane overlaying the acrosome region by immunoaggregation of P12 on mouse sperm. Further, we compared the immunoreactivity of P12 antibody to ten P12 variants, including six single-site mutated mutants (R19L, Y21V, D22G, R43G, K44S, and R45T), two multisite mutated mutants (R43G/K44S/R45T and L50H/R52G/K53A), and two deletion mutants (Nd10 and Cd8) in which 10 and 8 residues were deleted from the N- and C-terminals, respectively.