Cell-based cytotoxicity assays for engineered nanomaterials safety screening: exposure of adipose derived stromal cells to titanium dioxide nanoparticles.

BACKGROUND: Increasing production of nanomaterials requires fast and proper assessment of its potential toxicity. Therefore, there is a need to develop new assays that can be performed in vitro, be cost effective, and allow faster screening of engineered nanomaterials (ENMs). RESULTS: Herein, we report that titanium dioxide (TiO2) nanoparticles (NPs) can induce damage to adipose derived stromal cells (ADSCs) at concentrations which are rated as safe by standard assays such as measuring proliferation, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) levels. Specifically, we demonstrated that low concentrations of TiO2 NPs, at which cellular LDH, ROS, or proliferation profiles were not affected, induced changes in the ADSCs secretory function and differentiation capability. These two functions are essential for ADSCs in wound healing, energy expenditure, and metabolism with serious health implications in vivo. CONCLUSIONS: We demonstrated that cytotoxicity assays based on specialized cell functions exhibit greater sensitivity and reveal damage induced by ENMs that was not otherwise detected by traditional ROS, LDH, and proliferation assays. For proper toxicological assessment of ENMs standard ROS, LDH, and proliferation assays should be combined with assays that investigate cellular functions relevant to the specific cell type.

A novel GFP reporter mouse reveals Mustn1 expression in adult regenerating skeletal muscle, activated satellite cells and differentiating myoblasts.

AIM: Mustn1 has been implicated in myofusion as well as skeletal muscle growth and repair; however, the exact role and spatio-temporal expression of Mustn1 have yet to be fully defined. METHODS: Transgenic mice were generated with a 1512-bp sequence of the Mustn1 promoter directing the expression of GFP (Mustn1(PRO) -GFP). These mice were used to investigate the spatio-temporal expression of Mustn1(PRO) -GFP during skeletal muscle development and adult skeletal muscle repair, as well as various phases of the satellite cell lifespan (i.e. quiescence, activation, proliferation, differentiation). RESULTS: Mustn1(PRO) -GFP expression was observed within somites at embryonic day 12 and developing skeletal muscles at embryonic day 15 and 18. While uninjured adult tibialis anterior muscle displayed no detectable Mustn1(PRO) -GFP expression, cardiotoxin injury robustly elevated Mustn1(PRO) -GFP expression at 3 days post-injury with decreasing levels observed at 5 days and minimal, focal expression seen at 10 days. The expression of Mustn1(PRO) -GFP at 3 days post-injury consistently overlaid with MyoD although the strongest expression of Mustn1(PRO) -GFP was noted in newly formed myotubes that were expressing minimal levels of MyoD. By 5 days post-injury, Mustn1(PRO) -GFP overlaid in all myotubes expressing myogenin although cells were present expressing Mustn1(PRO) -GFP alone. The expression patterns of Mustn1(PRO) -GFP in regenerating muscle preceded the expression of desmin throughout the regenerative time course consistent with Mustn1 being upstream of this myogenic protein. Further, quiescent satellite cells located on freshly isolated, single myofibers rarely expressed Mustn1(PRO) -GFP, but within 24 h of isolation, all activated satellite cells expressed Mustn1(PRO) -GFP. Expression of Mustn1(PRO) -GFP in primary myoblasts diminished with prolonged time in proliferation media. However, in response to serum withdrawal, the expression of Mustn1(PRO) -GFP increased during myofusion (day 2) followed by declining expression thereafter. CONCLUSION: Mustn1(PRO) -GFP is expressed in activated satellite cells and myoblasts but continued time in proliferation media diminished Mustn1(PRO) -GFP expression. However, myoblasts exposed to serum withdrawal increased Mustn1(PRO) -GFP expression consistent with its demonstrated role in myofusion. The in vivo expression pattern of Mustn1 observed in regenerating and developing skeletal muscle is consistent with its presence in satellite cells and its critical role in myofusion.

Integrin alpha2beta1 affects mechano-transduction in slowly and rapidly adapting cutaneous mechanoreceptors in rat hairy skin.

The role of a transmembrane protein, integrin alpha2beta1, to modulate the neural responses of cutaneous mechanoreceptors to mechanical indentation was examined using an isolated skin-nerve preparation in a rat model. Skin and its intact innervation were harvested from the medial thigh of the hindlimb and placed in a dish containing synthetic interstitial fluid. Using a standard teased nerve preparation, the neural responses of single slowly or rapidly adapting mechanoreceptors (SA or RA, respectively) were identified and the afferents categorized according to standard protocols (i.e. response to constant stimuli). The most sensitive spot of a mechanoreceptor's receptive field was identified and then stimulated using controlled compressive stress (constant or dynamic loads between threshold and saturation load for SAs and RAs, respectively). Loads were applied before, during, and after passive diffusion into the skin of a function-blocking anti-integrin alpha2 monoclonal antibody (FBmAb) or one of two types of control antibodies (immunoglobulin G or a FBmAb conjugated with a secondary antibody). The sensitivities of both SA and RA mechanoreceptors were profoundly reduced in the presence of the FBmAb, while not changing the waveforms of their action potentials or their adaptation properties. Both control antibodies had no significant effect on mechanoreceptors' sensitivities. Following removal of the FBmAb, the effects in some neurons were partially reversible. Taken together, the data from this study support the hypothesis that integrin alpha2beta1 plays a significant role in modulating mechanoreceptive response to compressive indentation.

Activation of the transcription factor HIF-1 and its target genes, VEGF, HO-1, iNOS, during fracture repair.

One of the immediate sequelae of bone fracture is regional hypoxia resulting from vasculature disruption. Hypoxia stabilizes and activates the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha), which ultimately leads to HIF-1-regulated gene expression. Because nothing is known about HIF-1 involvement in bone regeneration, we performed a series of experiments to elucidate the expression pattern of HIF-1alpha and selected HIF-1 target genes using a rat femoral fracture model. Callus samples were obtained on postfracture days (PFD) 3, 5, 7, 10, 14, and 21. Quantitative RT-PCR (qRT-PCR) was employed to quantify the temporal mRNA expression patterns of HIF-1alpha, vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1). Elevated HIF-1alpha and VEGF expression was seen at all time points, with peak increases of approximately 6- and 2-fold relative to the intact bone present on PFD 10 for HIF-1alpha and VEGF, respectively. Robust activation of iNOS was detected solely on PFD 10 (6.8-fold) with all other time points showing slight downregulation. HO-1 expression peaked on PFD 3 (4.5-fold) with no significant changes on any other PFD. Western blot analysis verified the temporal expression patterns with HIF-1alpha protein expression showing a steady rise to a PFD 10 peak of approximately 18-fold. Similarly, the expression patterns for VEGF and HO-1 showed increases of approximately 4-fold at their PFD 10 and PFD 3 peaks, respectively. Immunohistochemical analysis of PFD 10 callus sections revealed coexpression of HIF-1alpha and VEGF in proliferating chondrocytes and active osteoblasts. Immunostaining for HO-1 on PFD 3 callus sections demonstrated strong expression in hematoma macrophages and vascular endothelial cells. Taken together, these experiments demonstrate for the first time that HIF-1alpha is upregulated at both transcriptional and translational levels in the fracture callus and indicate that PFD 10 may be a key angiogenic time point in the developing rat fracture callus.

Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers.

The present work utilizes electrospinning to fabricate synthetic polymer/DNA composite scaffolds for therapeutic application in gene delivery for tissue engineering. The scaffolds are non-woven, nano-fibered, membranous structures composed predominantly of poly(lactide-co-glycolide) (PLGA) random copolymer and a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer. Release of plasmid DNA from the scaffolds was sustained over a 20-day study period, with maximum release occurring at approximately 2 h. Cumulative release profiles indicated amounts released were approximately 68-80% of the initially loaded DNA. Variations in the PLGA to PLA-PEG block copolymer ratio vastly affected the overall structural morphology, as well as both the rate and efficiency of DNA release. Results indicated that DNA released directly from these electrospun scaffolds was indeed intact, capable of cellular transfection, and successfully encoded the protein beta-galactosidase. When tested under tensile loads, the electrospun polymer/DNA composite scaffolds exhibited tensile moduli of approximately 35 MPa, with approximately 45% strain initially. These values approximate those of skin and cartilage. Taken together, this work represents the first successful demonstration of plasmid DNA incorporation into a polymer scaffold using electrospinning.

Skeletal adaptation to mechanical stimuli in the absence of formation or resorption of bone.

Too often, unique loading environments fail to alter bone mass and morphology, calling to question the validity of Wolff's Law; the skeleton's plasticity to mechanical signals(1). We propose that bone can accommodate new loading environments without the need to form or resorb tissue, and that a critical aspect of bone tissue's ability to adapt to mechanical stimuli is first achieved via the plasticity of the osteocyte. We suggest that the osteocyte is capable of "normalizing" its local mechanical environment by modulating its cytoskeletal architecture, attachment to the matrix, configuration of the periosteocytic space, and communication channels to surrounding cells. We believe that through this local adaptive mechanism the osteocyte can accommodate the majority of changes in the mechanical milieu without altering the tissue architecture. It is only when bone tissue is subject to more severe (albeit rare) increases or decreases in the functional environment, the osteocyte participates in the formation and/or resorption of bone by coordinating site-specific recruitment of osteoblasts and/or osteoclasts. In vivo models of bone adaptation, combined with in situ reverse transcriptase-PCR, semi-quantitative RT-PCR, Northern analysis, immuno-cytochemistry and histomorphometry, can demonstrate how distinct mechanical stimuli influence the osteocyte's cytoskeletal and lacunar architecture, coupling (and uncoupling) of the osteocyte to the matrix and neighboring cells, and the osteocyte's participation in the recruitment and differentiation of osteoblasts and osteoclasts. Thus, the osteocyte controls three strategies to modulate its local and global environment in response to three distinct functional stimuli: 1) exogenous mechanical stimuli which are distinct from normal but sufficient to maintain bone mass, 2) mechanical stimuli which are osteogenic, and 3) disuse. If it is true that the resident cell population is capable of accommodating subtle changes in the functional milieu before modification of tissue morphology is deemed necessary, a novel strategy for the development of prophylaxes for osteopenia, osseointegration and fracture healing may become apparent.

Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation.

Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.

The E11 osteoblastic lineage marker is differentially expressed during fracture healing.

Given the complexity of the fracture healing process and the involvement of a multitude of cells, we hypothesize that a very large number of genes would be transcriptionally regulated during the repair process. To identify genes that play a role during bone regeneration in cellular events, such as proliferation, migration, adhesion, and differentiation, we employed differential display and compared mRNA populations isolated from postfracture (PF) day 3 calluses to those of intact (contralateral) rat femurs. One such gene whose expression was upregulated at PF day 3 is identified as the osteoblastic lineage marker, E11 antigen. E11 is a cell membrane protein localized predominantly on osteoblasts and osteocytes. In this study we show that E11 mRNA expression is consistently upregulated during fracture repair, with elevated levels (tenfold) seen as early as PF day 3. These high levels of expression were maintained throughout all of the early stages of repair examined (PF day 3-21 calluses) and paralleled those of another osteoblastic marker, osteopontin. Similarly, high protein levels were detected throughout the reparative phase of the callus, particularly in osteoblasts, preosteocytes, and osteocytes, localized within the newly made osteoid. No labeling was detected in fibroblasts, proliferating chondrocytes, or hypertrophic chondrocytes, consistent with previous results. Taken together, these results suggest that the E11 antigen is indeed a suitable marker for both osteoblasts and osteocytes and that it plays a significant role in osteogenesis.

Differential phosphorylation of paxillin in response to surface-bound serum proteins during early osteoblast adhesion.

An early signaling event during the adhesion and spreading of cells is integrin-mediated tyrosine phosphorylation of the cytoskeletal adaptor protein paxillin and the non-receptor tyrosine kinase pp125(FAK) at focal contacts. To determine the influence of surface-charge and -adsorbed adhesion proteins on this signaling pathway, paxillin phosphorylation was examined during attachment of MC3T3-E1 osteoblast-like cell onto charged and uncharged polystyrene, and on adsorbed layers of serum proteins, fibronectin (Fn), vitronectin (Vn), a mixture of Fn and Vn, and albumin. Paxillin phosphorylation was induced 2.4-fold (P < 0.05) on charged vs uncharged polystyrene only in the presence of serum proteins. Activation of paxillin via Fn or Vn alone, or in combination, resulted in significantly lower phosphorylation signals compared to whole serum (41 +/- 6.9%, P < 0.05, 45 +/- 5.9%, P < 0.05, and 76 +/- 9.8%, P < 0.075, respectively). Confocal laser microscopy confirmed increased co-localization of phosphotyrosine and paxillin at protruding lamellopodia of spreading osteoblasts on charged vs uncharged serum-pretreated polystyrene. Taken together, these data suggest that subtle differences in surface characteristics mediate effects on adhering cells via adsorbed serum proteins involving the cytoskeletal adaptor protein paxillin.

Expression of integrin alpha2beta1 in axons and receptive endings of neurons in rat, hairy skin.

Integrin alpha2beta1 has been considered as a mechano-chemical transducer in endothelial and muscle cells. However, little data is available to show whether integrins play a role in the process of mechanical transduction in peripheral mechanosensory neurons. Using immunohistochemistry, we demonstrate that cutaneous neurons express the extracellular matrix (ECM) receptor integrin alpha2beta1. Specifically, we show that integrins alpha2 and beta1 are co-localized with peripherin in the receptive endings of cutaneous neurons in rat, hairy skin. Integrin immunofluorescence was minimal along the axons of large diameter neurons. These results, together with findings by other investigators, provide evidence suggesting that integrin alpha2beta1 may be a linking agent between mechanical stress in the ECM and modulation of the neuronal response of mechanically sensitive neurons.

Temporal expression of the chondrogenic and angiogenic growth factor CYR61 during fracture repair.

The repair of a fractured bone is a complex biological event that essentially recapitulates embryonic development and requires the activity of a number of different cell types undergoing proliferation, migration, adhesion, and differentiation, while at the same time expressing a host of different genes. To identify such genes, we employed differential display and compared messenger RNA (mRNA) populations isolated from postfracture (PF) day 5 calluses to those of intact rat femurs. One such gene in which expression was up-regulated at PF day 5 is identified as CYR61, a member of the CCN family of secreted regulatory proteins. CYR61 is a growth factor that stimulates chondrogenesis and angiogenesis. We show that its mRNA expression during fracture repair is regulated temporally, with elevated levels seen as early as PF day 3 and day 5, rising dramatically at PF day 7 and day 10, and finally declining at PF day 14 and day 21. At the highest peak of expression (PF day 7 and day 10, which correlates with chondrogenesis), CYR61 mRNA levels are approximately 10-fold higher than those detected in intact femurs. Similarly, high protein levels are detected throughout the reparative phase of the callus, particularly in fibrous tissue and periosteum, and in proliferating chondrocytes, osteoblasts, and immature osteocytes. The secreted form of CYR61 also was detected within the newly made osteoid. No labeling was detected in hypertrophic chondrocytes or in mature cortical osteocytes. These results suggest that CYR61 plays a significant role in cartilage and bone formation and may serve as an important regulator of fracture healing.

Increased expression of matrix metalloproteinase-1 in osteocytes precedes bone resorption as stimulated by disuse: evidence for autoregulation of the cell's mechanical environment?

An in vivo animal model of bone adaptation was used to examine a possible role for matrix metalloproteinase-1 in the local mediation of bone remodeling: to corrode the coupling of osteocytes to the matrix in an attempt to autoregulate the cell's perception of its mechanical environment. Twelve young (12-16 months old) skeletally mature turkeys were separated into groups to be studied for stimulus periods of either 3 or 30 days. In each animal, the left ulna was functionally isolated and subjected to either disuse or 3,000 microstrain at 1 Hz for 10 minutes per day. The right ulna remained intact and served as an intra-animal control. No significant differences in bone area were detected at 3 days; however, ulnae subjected to disuse lost 8 +/- 4% (+/-SD) of bone area by 30 days. Over the same period, ulnae subjected to the mechanical stimulus gained 21 +/- 9% of bone area. With use of in situ reverse transcription-polymerase chain reaction, less than 2% of the osteocytes examined from the intact control ulnae stained positively for matrix metalloproteinase-1 mRNA. An antibody raised against matrix metalloproteinase-1 revealed no positively labeled osteocytes in the intact ulnae. This low percentage of osteocytes expressing matrix metalloproteinase-1 mRNA was similar to that seen in ulnae subjected to the osteogenic mechanical stimuli. In contrast, ulnae subjected to either 3 or 30 days of disuse showed evidence of matrix metalloproteinase-1 mRNA activity in a high percentage of osteocytes (89 +/- 5 and 66 +/- 8%, respectively; each time point significantly different from intact ulnae, as well as from each other, p < 0.05). The percentage of osteocytes labeled with the anti-matrix metalloproteinase-1 antibody was also highly elevated following 3 days of disuse (74 +/- 17%). These data demonstrate that an early response of bone to disuse is the upregulation of matrix metalloproteinase-1 activity in osteocytes. It is proposed that this upregulation of collagenase activity is indicative of the cell's degradation of coupling to the matrix, and it thus reflects the osteocyte's regulation of its own mechanical environment. We believe that such autoregulation of the osteocyte's physical environment will accommodate subtle changes in the bone's functional environment without the need to add or resorb bone tissue.

Heat shock proteins in retinal neurogenesis: identification of the PM1 antigen as the chick Hsc70 and its expression in comparison to that of other chaperones.

While the role of heat shock proteins under experimental stress conditions is clearly characterized, their expression in unstressed cells and tissues and their functions in normal cell physiology, besides their chaperone action, remain largely undetermined. We report here the identification in chicken of the antigen recognized by the monoclonal antibody PM1 [Hernández-Sánchez et al. (1994) Eur. J. Neurosci., 6,1801-1810] as the noninducible chaperone heat-shock cognate 70 (Hsc70). Its identity was determined by partial peptide sequencing, immuno-crossreactivity and two-dimensional gel-electrophoresis. In addition, we examined its expression during chick embryo retinal neurogenesis. The early widespread Hsc70 immunostaining corresponding to most, if not all, of the neuroepithelial cells becomes restricted to a subpopulation of these cells in the peripheral retina as development proceeds. On the other hand, retinal ganglion cells, differentiating in the opposite central-to-peripheral gradient, retained Hsc70 immunostaining. Other molecular chaperones, the heat-shock proteins Hsp40, Hsp60 and Hsp90, did not seem to compensate the loss of Hsc70. They also showed decreasing immunostaining patterns as neurogenesis proceeds, although distinctive from that of Hsc70, whereas Hsp70 was not detected in the embryonic retina. This precise cellular and developmental regulation of Hsc70, a generally considered constitutive molecular chaperone, in unstressed embryos, together with the expression of other chaperones, provides new tools and a further insight on neural precursor heterogeneity, and suggests possible specific cellular roles of chaperone function during vertebrate neurogenesis.

Cloning of a novel cDNA expressed during the early stages of fracture healing.

Using differential mRNA display (DD-PCR), a novel cDNA, FxC1 (Fracture Callus 1) was isolated from the early stages of a healing fractured femur. Utilizing 5' RACE PCR, a 598-bp full-length cDNA was obtained for FxC1 that contains an open reading frame (ORF) of 243 bp, encoding for an 80 amino acid protein. Within this ORF, a leucine zipper motif was present. In vitro transcription/translation of the full-length cDNA generated the expected 9-kDa protein. Northern analysis reveals that this gene is expressed in calluses harvested from post-fracture day 5, 7 and 10, as well as in several other tissues and bone-derived cell lines. During the differentiation of MC3T3 cells along the osteoblast lineage, FxC1 expression increases 3- to 4-fold during the production and deposition of matrix proteins, suggesting a possible role for this protein in cell differentiation.

Enhancement of fracture healing by low intensity ultrasound.

Fracture healing is a highly complex regenerative process that is essentially a replay of developmental events. These events include the action of many different cell types, a myriad of proteins, and active gene expression that in the majority of cases ultimately will restore the bone's natural integrity. Several biologic and biophysical approaches have been introduced to minimize delayed healing and nonunions, some with promising results. One example of such an approach is low intensity pulsed ultrasound, a noninvasive form of mechanical energy transmitted transcutaneously as high frequency acoustical pressure waves in biologic organisms. Numerous in vivo animal studies and perspective double blind placebo controlled clinical trials have shown that low intensity ultrasound is capable of accelerating and augmenting the healing of fresh fractures. Preliminary evidence suggests efficacy in the treatment of delayed healing and nonunions as well. This article reviews the animal and clinical studies that consider the effects of ultrasound on fracture healing, and the in vivo and in vitro work that strives to identify the biologic mechanism(s) responsible for the ultrasound induced enhancement of osteogenesis and fracture healing.

An antibody to the tetraspan membrane protein CD9 promotes neurite formation in a partially alpha3beta1 integrin-dependent manner.

The tetraspan cell surface glycoprotein, CD9, has been implicated in cellular signaling during growth and differentiation in the hematopoietic and nervous systems. Because CD9 expression is induced early in development in sensory and sympathetic neuroblasts, we investigated the role of CD9 in neurite outgrowth. We plated dissociated cells from neonatal sympathetic ganglia on immobilized anti-CD9 antibodies or antibodies against other cell surface molecules. We show here that B2C11, an anti-CD9 antibody that has been shown previously to activate Schwann cells in vitro, promotes robust neurite outgrowth from sympathetic neurons that is greater than that on other antibody surfaces and is comparable to neurite outgrowth on a collagen substratum. In addition, B2C11 causes dramatic morphological changes in neurons and glia from dissociated ganglia, including a flattening of these cells. Because CD9 interacts with integrins in many cell types including Schwann cells, and specifically with the alpha3beta1 integrin in some cells, we tested whether the effect of B2C11 on neurite outgrowth is mediated by this integrin. An anti-alpha3beta1 antibody, Ralph 3-1, attenuates the extent of neurite outgrowth on B2C11 and collagen, but not on laminin. Because the alpha3beta1 integrin has been shown to mediate neurite outgrowth on different substrates, these results provide a functional significance for the CD9-alpha3beta1 interaction; downstream signaling may be activated by this cis interaction on the cell surface in response to external cues that promote neurite outgrowth.

Association of the tetraspan protein CD9 with integrins on the surface of S-16 Schwann cells.

The cell surface glycoprotein CD9 is a member of a group of proteins known as the "tetraspan" or "transmembrane 4 superfamily." Previous work with non-neural cells has shown that CD9 associates in cis with integrins and small GTP-binding proteins on the cell surface. To extend our recent findings showing that perturbation of CD9 alters Schwann cell adhesion, proliferation, and migration, as well as neurite outgrowth in sympathetic neurons, we have searched for CD9-associated proteins in S-16 Schwann cells. We demonstrate here that CD9 is specifically coprecipitated from S-16 cell extracts by antibodies against integrins alpha 3, alpha 6, and beta 1. In addition, double immunofluorescence labeling and co-capping experiments indicate that CD9 is specifically co-localized with these integrins on the cell membrane of S-16 Schwann cells.

CD9, a major platelet cell surface glycoprotein, is a ROCA antigen and is expressed in the nervous system.

We previously generated a monoclonal antibody (mAb), ROCA1, which binds preferentially to rostral versus caudal sympathetic ganglia and intercostal nerves. Two other mAbs, ROCA2 and B2C11, bind to the same structures but not in rostrocaudal gradients. All three mAbs recognize a 26 kDa cell surface protein. Amino acid sequence data obtained from the affinity purified 26 kDa protein showed some homology with human CD9, a tetraspan protein implicated in intercellular signaling in hematopoietic cells. Using the PCR, we obtained cDNA clones representing the entire rat CD9 coding sequence from sciatic nerve and sympathetic ganglia. ROCA1, ROCA2, and B2C11 each immunoprecipitate a 26 kDa protein from CHO cells stably transfected with one of the clones, demonstrating that the ROCA cell surface antigen is indeed rat CD9. We find that CD9 mRNA is widely expressed, with particularly high levels present in a number of neural tissues. In situ hybridization demonstrates that peripheral neurons and Schwann cells, as well as adrenal chromaffin cells express CD9 mRNA. Consistent with immunoblot analyses showing that, unlike the ROCA1 epitope, the 26 kDa protein is not expressed in a rostrocaudal gradient, we find similar levels of rat CD9 mRNA in rostral and caudal intercostal nerves. In developing postnatal rat sciatic nerve, CD9 mRNA levels are coordinately regulated with the expression of myelin genes. These results provide another example of a cell surface protein expressed by both hematopoietic and neural cells, and suggest a role for CD9 in intercellular signaling in the nervous system.

An anti-CD9 monoclonal antibody promotes adhesion and induces proliferation of Schwann cells in vitro.

We have recently found that CD9, a cell surface glycoprotein involved in intercellular signaling in hematopoietic cells, is also expressed by neurons and glia in the peripheral nervous system. Antibody perturbation experiments were conducted to examine the function of CD9 in neural cells. Three anti-CD9 monoclonal antibodies (mAbs) (ROCA1, ROCA2, B2C11) were tested for their ability to promote adhesion of several Schwann cell lines (S-16, RN22, JS1), primary Schwann cells and PC12 cells. Only B2C11 promotes adhesion in all cells tested. Although ROCA2 immunolabels living cells strongly, it had no effect on the adhesion of any of these cells. In addition, ROCA1 and several positive-staining, control mAbs also had no effect. Another mAb, 192-IgG, directed against the low affinity NGF receptor, also promotes the adhesion of S-16, PC12, and primary Schwann cells. In addition to adhesion, contact of S-16 Schwann cells with B2C11 specifically induces morphological changes and robust proliferation. None of the other mAbs, including 192-IgG, induce proliferation of S-16 cells. These results provide evidence that CD9 may be involved in signaling, activation and growth regulation of cells in the nervous system.