Bone morphogenetic protein-5 (BMP-5) promotes dendritic growth in cultured sympathetic neurons
© Beck et al 2001
Received: 20 July 2001
Accepted: 11 September 2001
Published: 11 September 2001
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© Beck et al 2001
Received: 20 July 2001
Accepted: 11 September 2001
Published: 11 September 2001
BMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons.
Sympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG) during times of initial growth and rapid expansion of the dendritic arbor.
These data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.
Bone morphogenetic proteins (BMPs) are secreted signaling molecules of the TGF-β superfamily that have been implicated in the control of a host of critical developmental phenomena in the central and peripheral nervous systems [1–3]. BMP-5, one of the more prominently expressed BMPs in the nervous system, has been detected in multiple regions of the nervous system throughout development and into adulthood [3–6], yet its biological activities in the nervous system are not well defined. A role for BMP-5 in dorsal forebrain patterning has been proposed based on its expression in the dorsal midline of the developing forebrain and observations that ectopic expression of BMP-5 in the developing neural tube of chicks markedly downregulates ventral markers while maintaining dorsal markers [5, 7]. Further support for BMP-5 regulation of early forebrain development has been provided by studies of Bmp5/Bmp7 double mutants . However, reports that BMP-5 in the mouse brain exhibits peak expression levels in the adult striatum and brainstem and that maximal expression in the hippocampus and cerebellum occurs at E18 through PN1 and again in the mature nervous system , suggest additional roles for BMP-5 during later stages of neural development and into adulthood.
BMPs have been divided into subgroups based on structural and evolutionary considerations . Although closely related BMPs have been shown to elicit distinct cellular responses [5, 9–13], members within a subgroup often display conservation of not only structure, but also function [4–6, 14]. BMP-5 belongs to the 60A subgroup of BMPs, which also includes BMP-6/Vgr-1, BMP-7/OP-1, BMP-8a/OP-2, BMP-8b and Drosophila 60A [3, 8]. Other members of the 60A subgroup have been shown to modulate neuronal morphogenesis through selective effects on dendrites. Thus, BMPs 6, 7, and 60A stimulate dendritic growth in cultured sympathetic neurons derived from either perinatal or adult ganglia in the absence of effects on cell survival or axonal growth [15–17]. BMP-7 has also been shown to enhance dendritic growth in hippocampal, cortical and spinal motor neurons [18–20].
Whether BMP-5 similarly promotes dendritic growth has not been previously addressed. Since dendrites are the primary site of synapse formation, we felt it was important to examine this possibility. Moreover, since dendritic remodeling occurs throughout the life of the animal, such studies could suggest a function for BMP-5 in the adult nervous system. In this report, we demonstrate that like other members of the 60A subgroup, BMP-5 triggers robust dendritic growth in sympathetic neurons in vitro coincident with activation of Smad-1. Noggin and follistatin, soluble proteins known to function as physiological antagonists for BMP-7 , also inhibit the dendrite-promoting activity of BMP-5. Furthermore, BMP-5 mRNA and protein are detected in intact sympathetic ganglia and neuron/glia cocultures, respectively, consistent with a proposed role for BMP-5 in regulating dendritic growth in sympathetic neurons in vivo.
BMP-5 is widely expressed in the nervous system throughout development and into adulthood [3–6], yet the only function described for this growth factor thus far is dorsal patterning of the developing forebrain [5–7]. Our data suggest that BMP-5 may also regulate later stages of neural development, specifically dendritic morphogenesis. The most direct support for this hypothesis is the finding that addition of BMP-5 to sympathetic neurons in culture causes these cells to extend multiple dendritic processes. These data are consistent with conclusions from previous studies indicating that dendrite-promoting activity is restricted to BMPs from the 60A or dpp (BMPs 2 and 4) subgroups and is not observed with BMPs from other subgroups such as BMP-3, BMP-13 or dorsalin, or with other members of the TGF-β superfamily such as activin, TGF-β1, -β2 or -β3 [16, 17]. Functional redundancy between BMPs of the 60A subgroup has been previously reported with respect to other developmental endpoints , of which some, such as upregulation of cell adhesion molecules , may be directly relevant to effects on dendritic growth. BMP-6 and BMP-7 as well as dpp subgroup members, BMP-2 and BMP-4, have been shown to influence other aspects of sympathetic neuron development, such as differentiation of adrenergic sympathetic neurons from neural crest [13, 31–38] and neuropeptide phenotype . It will be of interest to determine if BMP-5 also exhibits functional redundancy with respect to these effects.
Pharmacological studies of BMP-5 indicate that relative to BMP-7, BMP-5 is less potent but equally efficacious in promoting dendritic growth in cultured sympathetic neurons. These data, together with observations that maximally effective concentrations of BMP-5 and BMP-7 are not additive, suggest that the two ligands share aspects of a common signaling pathway. It has been shown that phosphorylation of Smad-1 precedes dendritic extension in cultured sympathetic neurons exposed to BMP-7; moreover, expression of a dominant negative construct of Smad-1 in cultured sympathetic neurons significantly inhibits BMP-7-induced dendritic growth in these neurons . These data suggest that activation of Smad-1 is a necessary component of the signal transduction pathway by which BMP-7 induces dendritic growth. In this report we demonstrate that BMP-5 similarly induces Smad-1 phosphorylation in sympathetic neurons as detected by Western blot analyses using antibodies specific for the phosphorylated form of Smad-1. These data suggest conserved mechanisms of signaling within the 60A subgroup. The molecular mechanism(s) of BMP-induced dendritic growth downstream of Smad-1 activation are not well characterized. Previous studies have demonstrated that transcriptional and translational events are required for dendritic growth in response to BMPs , but the gene expression profile responsible for BMP-induced dendritic growth has yet to be determined. Thus, it is not clear if BMP-5 or -7 induces dendritic growth directly, or if some other factor made by the cells in response to BMPs is responsible for initiating dendritic growth.
BMPs activate Smad-1 by binding to type I and type II serine-threonine kinase receptors [40, 41]. Specific receptor subunits shown to bind BMPs include BMP receptor type IA (BMPR-IA), BMPR-IB, BMR-II, activin receptor type I (ActR-I), and ActR-II [42–44]. BMP ligands can bind to either type I or type II receptor subunits independently, but both receptor types are required for high-affinity binding and signaling . The finding that the soluble BMPR-IA-Fc chimera significantly inhibits BMP-5 induced dendritic growth suggests that BMP-5 may be activating the Smad-1 signaling pathway via interactions with BMPR-IA. Although the physiological relevance of this finding has yet to be confirmed by ligand binding studies using endogenous neuronal BMPR-IA, these data are consistent with reports that BMPR-IA is the predominant BMP receptor type expressed in embryonic and postnatal superior cervical ganglia (SCG) .
It is becoming increasingly evident that BMP signaling is regulated not only by the spatiotemporal expression of BMP ligands and receptors, but also by relative levels of soluble BMP antagonists, which directly bind BMPs and prevent functional receptor/ligand interaction [1, 24–28]. The different BMP antagonists bind to BMPs and other TGF-β family members with varying degrees of specificity. For example, follistatin binds both activin and BMP-7 avidly but competes weakly or not at all with the type I receptor for BMP-4 binding [25, 45], whereas noggin binds to BMPs -2 and -4 with greater affinity than BMP-7 . Whether BMP-5 function can be antagonized by noggin or follistatin has not been previously reported, but our results suggest that simultaneous addition of either antagonist with BMP-5 significantly inhibits the dendrite-promoting activity of BMP-5 in a concentration-dependent manner. Thus, profiling the BMP binding affinities as well as the expression patterns of these BMP antagonists will be critical to understanding the regulation of BMP-5 signaling in the nervous system.
If BMP-5 is important in regulating dendritic growth in intact ganglia, its expression should correlate with periods of dendritic growth in vivo. In sympathetic ganglia, dendritic growth begins prenatally and maximal expansion of the dendritic arbor occurs during postnatal weeks 1 and 2 [47, 48]. RT-PCR analyses of intact SCG indicate that cells of the SCG express BMP-5 transcripts from E20 through P7. Preliminary observations indicate that these BMP-5 transcripts are translated into protein in SCG in vivo as assessed by Western blot analyses using BMP-5 Ab (P. Lein, unpublished observations). Earlier studies have demonstrated the expression of BMP-4 transcripts in developing avian sympathetic ganglia , suggesting the presence of multiple BMPs in sympathetic ganglia throughout development. These data, in conjunction with observations that mRNA for BMP type IA and type II receptors are expressed in the developing sympathetic ganglia , are consistent with a potential role for BMP-5 in regulating the initiation and rapid expansion of the dendritic arbor in sympathetic ganglia of perinatal animals.
The cellular distribution of BMP-5 was determined by immunocytochemistry in sympathetic neurons cocultured with ganglionic glial cells. Both neurons and glial cells express BMP-5 protein. In situ hybridization analyses of BMP-6 and BMP-7 indicate that both cell types also express BMP transcripts (P. Lein, unpublished observations). These findings are consistent with previous reports that dendritic growth can be induced in sympathetic neurons in vitro when cultured at high neuronal cell density  or in the presence of ganglionic glial cells .
Dendritic growth continues, albeit to a lesser extent, into adulthood, and dendritic remodeling occurs throughout the animal's life. Mature sympathetic neurons cultured from adult animals respond to BMP-7 with enhanced dendritic growth , and treatment with BMP-7 enhances recovery in animal models of stroke [52–56]. Although BMP-5 mRNA was not detected in adult SCG, Western blot analyses indicate that BMP-5 protein is present in adult SCG (P. Lein, unpublished observations), presumably derived from nonganglionic sources such as serum or target tissues. These observations together with reports that BMP-5 is expressed at significant levels in the adult nervous system  suggest a potential role for BMP-5 in modulating dendritic morphology not only during development, but also in adult nervous systems.
These data suggest that BMP-5 regulates dendritic growth. Addition of BMP-5 to sympathetic neurons in culture triggers significant dendritic growth that is concentration-dependent. Data from western blot analyses using Ab specific for phosphorylated epitopes of Smad-1 as well as analyses of dendritic growth in cultures exposed to BMP-5 in the presence of a soluble BMPR-IA-Fc chimeric protein are consistent with a signaling pathway that involves binding to the BMPR-IA and activation of Smad-1. BMP-5 signaling may be modulated by noggin and follistatin since these BMP antagonists were observed to inhibit the dendrite-promoting activity of BMP-5. Spatiotemporal patterns of BMP-5 expression at the mRNA level, as assessed by RT-PCR, and the protein level, as determined by immunocytochemistry correspond to periods of initial dendritic growth and rapid expansion of the dendritic arbor. These observations, together with previously published reports from other laboratories indicating significant levels of BMP-5 expression in the developing and adult nervous system  suggest a potential role for BMP-5 in modulating dendritic morphology not only during development, but also in adult nervous systems.
Purified human recombinant BMPs (5, 6 and 7) were prepared using previously published methods  and provided by Creative Biomolecules (Hopkinton, MA). Affinity-purified polyclonal antibody (Ab) specific for BMP-5, the blocking peptide for the BMP-5 Ab, and the recombinant human BMP-RIA-Fc chimera were purchased from Research Diagnostics (Flanders, NJ). Ab specific for the phosphorylated form of Smad-1 (Ser 463/465) as well as Ab that recognizes both phosphorylated and nonphosphorylated Smad-1 (e.g., total Smad-1) was purchased from Upstate Biological (Lake Placid, NY). Xenopus noggin protein  was the generous gift of Drs. Josè de Jesús and Richard Harland (UC at Berkeley). Recombinant human follistatin (B4384) was obtained through Dr. A.F. Parlow at the NHPP, NIDDK (Torrance, CA).
Sympathetic neurons were dissociated from the SCG of perinatal (E21 to PN1) Holtzmann rats (Harlan Sprague-Dawley, Rockford, IL) according to previously described methods . Cells were plated onto glass coverslips (for immunocytochemical and morphological studies) or 35 mm plastic culture dishes (for Western blot analyses) precoated with 100 μg/ml poly-D-lysine (Sigma, St. Louis, MO). Cultures were maintained in serum-free medium supplemented with β-NGF (100 ng/ml), bovine serum albumin (500 μg/ml), insulin (10 μg/ml), and transferrin (20 μg/ml). In most experiments, endogenous non-neuronal cells were eliminated from cultures by adding cytosine-β-D-arabinofuranoside (Sigma, St. Louis, MO) to the culture medium at 1 μM for 48 hr beginning on day 2. In some experiments this antimitotic was not added to cultures but rather endogenous non-neuronal cells were allowed to proliferate. Previous studies have demonstrated that under these culture conditions, the non-neuronal cells are primarily ganglionic glia .
Dendritic growth was assessed in cultures immunostained with monoclonal antibodies (mAb) previously shown to react selectively with the somatodendritic compartment of cultured sympathetic neurons . These mAb's include SMI-52, which is specific for the cytoskeletal protein MAP2, and SMI-32, which reacts with the non-phosphorylated forms of the M and H neurofilament subunits (Sternberger Immunocytochemicals, Baltimore, MD). Antigens were localized by indirect immunofluorescence as previously described . Dendritic growth was quantified using SPOT image analysis system. Data in the text are presented as the mean ± S.E.M. and statistical significance was determined using ANOVA followed by Tukey's test.
Western blot analyses were performed on purified recombinant BMPs to assess the specificity of the BMP-5 Ab, and on cell lysates of cultured sympathetic neurons to assess the effects of BMP-5 on phosphorylation of Smad1 as well as levels of total Smad (nonphosphorylated and phosphorylated). Cell lysates were obtained by rinsing 12-day old neuronal cell cultures with ice-cold phosphate-buffered saline prior to trituration in ice-cold lysis buffer (PBS supplemented with 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml PMSF and 300 μg/ml aprotinin). Cell lysates were microfuged at maximum speed for 5 min and the resultant supernatant collected. Protein concentration was determined using the Bradford assay (BioRad, Hercules, CA). Samples containing equivalent amounts of protein were resolved by 12% polyacrylamide SDS PAGE under reducing conditions and then electroblotted onto PVDF membranes. Blots were blocked at room temperature for 1 hour in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween-20) containing 5% dried fat-free milk, then incubated overnight at 4°C in TBS-T containing 0.5% milk and primary Ab (0.5 μg/ml for BMP-5 Ab; 10 μg/ml for Smad-1 Ab). Blots were washed twice with TBS-T containing 0.5% milk, then incubated at room temperature for 2 hours in TBS-T containing 0.5% milk and 1:5000 dilutions of secondary Ab conjugated to peroxidase (for BMP-5, anti-goat IgG-peroxidase from Chemicon, Temecula CA; for Smad1, anti-rabbit Ig-peroxidase from Amersham, Piscataway, NJ). Subsequently, blots were washed three times as described above, and visualized using an enhanced chemiluminescence detection method (ECL, Amersham). Blots of cell lysates were stripped and reprobed using antibodies specific for α-tubulin (1:10,000, Sigma). To quantify data, films were scanned using an HP ScanJet ADF scanner and HP Precision ScanPro software, and band density determined as arbitrary absorption units using the MacBas software program (version 2.31, Fuji Film).
BMP-5 Ab was used to localize BMP-5 protein in SCG cultures containing sympathetic neurons and ganglionic glial cells. After 2 weeks in culture, cells were fixed in 4% paraformaldehyde, permeabilized with methanol at -20°C (Sigma, St. Louis, MO), and then reacted with anti-BMP-5 Ab (10 μg/ml). Immunoreactivity was visualized by indirect immunofluorescence as previously described . The specificity of the immunoreaction was determined by preincubating the BMP-5 Ab with its specific blocking peptide (100 μg/ml) prior to reaction with the cultures.
Total RNA was extracted from freshly harvested superior cervical ganglia (SCG) using Trizol (Life Technology, Carlsbad, CA). RNA samples (5 μg) were reverse transcribed using random primers at annealing temperatures of 65°C (You-Prime-the-First-Strand kit, Amersham, Piscataway, NJ). Resultant cDNA was amplified by PCR for 35 cycles using an annealing temperature of 55°C for 30 sec and denaturing temperature of 95°C for 30 sec; the Mg++ concentration in these reactions was 1.5 mM. As a negative control, each sample was run through PCR without prior reverse-transcription. Primers used for amplification of BMP-5 cDNA were designed to unique sequences of rat BMP-5 using the Primer3 program http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi. The specific primer sequences were BMP-5 sense, 5'-TTATGCAAAAGGAGGCTTGG-3' and BMP-5 antisense, 5'-TCATGACCATGTCAGCATCA-3'. After synthesis, PCR products were subjected to 1% agarose gel electrophoresis and found to have the expected size of 420 base pairs.
bone morphogenetic protein
BMP receptor type IA
embryonic day 20 or 21
PN3 or PN7, postnatal day 1, 3 or 7
superior cervical ganglia
We thank Creative Biomolecules (Hopkinton, MA), now known as Curis (Cambridge, MA), for supplying the purified recombinant BMPs. This work was supported by a Johns Hopkins University School of Public Health Faculty Innovation Award (PJL), NSF grant #IBN 01–21210 (DH) and NIH grants HL54659 (DBJ) and HL61013 (DBJ).
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