Asef2 and Neurabin2 cooperatively regulate actin cytoskeletal organization and are involved in HGF-induced cell migration.

The tumor suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumors. APC interacts with the Rac1- and Cdc42-specific guanine-nucleotide exchange factors (GEF), Asef and Asef2, which contain an APC-binding region (ABR) in addition to Dbl homology, Pleckstrin homology (PH) and Src homology 3 (SH3) domains. APC stimulates the GEF activity of Asef and Asef2, and thereby regulates cell adhesion and migration. Here we show that Asef2, but not Asef, interacts with Neurabin2/Spinophilin, a scaffold protein that binds to Filamentous actin (F-actin). In response to hepatocyte growth factor (HGF) treatment of HeLa cells, Asef2, Neurabin2 and APC were induced to accumulate and colocalize in lamellipodia and membrane ruffles. Neurabin2 did not affect the GEF activity of Asef2. RNA interference experiments showed that Asef2, Neurabin2 and APC are involved in HGF-induced cell migration. Furthermore, knockdown of Neurabin2 resulted in the suppression of Asef2-induced filopodia formation. These results suggest that Asef2, Neurabin2 and APC cooperatively regulate actin cytoskeletal organization and are required for HGF-induced cell migration.

Follistatin inhibits the function of the oocyte-derived factor BMP-15.

Recent studies have highlighted the importance of a novel oocyte-derived growth factor, bone morphogenetic protein-15 (BMP-15) in the regulation of proliferation and differentiation of granulosa cells in the ovary. Namely, BMP-15 stimulates granulosa cell mitosis and inhibits follicle-stimulating hormone (FSH) receptor mRNA expression in granulosa cell, thereby playing a critical role in the elaborate mechanism controlling ovarian folliculogenesis. At present, however, nothing is known about molecules which may regulate the biological activity of BMP-15. Here we demonstrate evidence that follistatin can form an inactive complex with BMP-15, through which follistatin inhibits BMP-15 bioactivities. The binding of follistatin to BMP-15 was directly demonstrated by a surface plasmon resonance biosensor, and the ability of follistatin to inhibit BMP-15 functions was determined by established BMP-15 bioassays using primary rat granulosa cells. Specifically, follistatin attenuated BMP-15 stimulation of granulosa cell proliferation and reversed BMP-15 inhibition of FSH receptor mRNA expression leading to the suppression of FSH-induced progesterone synthesis. This is the first demonstration of the biochemical interaction and biological antagonism of follistatin and BMP-15, which may be involved in the complex yet well-controlled mechanism of the regulation of follicle growth and development.

Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kappaB ligand.

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.

Dorsal spinal cord inhibits oligodendrocyte development.

Oligodendrocytes are the myelinating cells of the mammalian central nervous system. In the mouse spinal cord, oligodendrocytes are generated from strictly restricted regions of the ventral ventricular zone. To investigate how they originate from these specific regions, we used an explant culture system of the E12 mouse cervical spinal cord and hindbrain. In this culture system O4(+) cells were first detected along the ventral midline of the explant and were subsequently expanded to the dorsal region similar to in vivo. When we cultured the ventral and dorsal spinal cords separately, a robust increase in the number of O4(+) cells was observed in the ventral fragment. The number of both progenitor cells and mature cells also increased in the ventral fragment. This phenomenon suggests the presence of inhibitory factor for oligodendrocyte development from dorsal spinal cord. BMP4, a strong candidate for this factor that is secreted from the dorsal spinal cord, did not affect oligodendrocyte development. Previous studies demonstrated that signals from the notochord and ventral spinal cord, such as sonic hedgehog and neuregulin, promote the ventral region-specific development of oligodendrocytes. Our present study demonstrates that the dorsal spinal cord negatively regulates oligodendrocyte development.

A functional bone morphogenetic protein system in the ovary.

Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.

Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo.

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.

Interaction between soluble type I receptor for bone morphogenetic protein and bone morphogenetic protein-4.

Bone morphogenetic proteins (BMPs) are multifunctional proteins that comprise the largest subfamily of the transforming growth factor-beta. These proteins bind to types I and II serine/threonine kinase receptors. Ligand-induced heteromeric dimerization of these receptors is the key event in initiation of biological responses. We report here large-scale expression and purification of extracellular domain of the type I receptor for BMP-2/4, using a silkworm expression system. This soluble form of BMP receptor (sBMPR) was in monomer form in solution and bound to BMP-4 but not to activin or transforming growth factor-beta1. Surface plasmon resonance studies showed that kinetic parameters of sBMPR for BMP-4 consisted of a relatively rapid association rate constant (ka = 3.81 +/- 0.19 x 10(4) s-1 M-1) and an extremely slow dissociation rate constant (kd = 3.69 +/- 0.26 x 10(-4) s-1). From these two kinetic parameters, affinity was determined to be similar to that of the intact membrane-associated receptor expressed on COS cells. sBMPR inhibited the alkaline phosphatase activity in BMP responsive cell lines such as mouse osteoblastic cell MC3T3-E1 and bone marrow stromal cell ST2. These data indicate that the extracellular domain of type I receptor for BMP-2 and BMP-4 is sufficient for high-affinity binding to its ligands and should prove useful in understanding the role of BMP-2/4 in vivo, because a suitable high-affinity anti-BMP antibody has yet to be developed.