Activated fibroblasts are key players in the injury response, tumorigenesis, fibrosis, and inflammation. Dichotomous outcomes in response to varied stroma-targeted therapies in cancer emphasize the need to disentangle the roles of heterogeneous fibroblast subsets in physiological and pathophysiological settings. In wound healing, fibrosis, and myriad tumor types, fibroblast activation protein (FAP) and alpha-smooth muscle actin (αSMA) identify distinct, yet overlapping, activated fibroblast subsets. Prior studies established that FAP reactive fibroblasts and αSMA myofibroblasts can exert opposing influences in tumorigenesis. However, the factors that drive this phenotypic heterogeneity and the unique functional roles of these subsets have not been defined. We demonstrate that a convergence of ECM composition, elasticity, and transforming growth factor beta (TGF-β) signaling governs activated fibroblast phenotypic heterogeneity. Furthermore, FAP reactive fibroblasts and αSMA myofibroblasts exhibited distinct gene expression signatures and functionality in vitro, illuminating potentially unique roles of activated fibroblast subsets in tissue remodeling. These insights into activated fibroblast heterogeneity will inform the rational design of stroma-targeted therapies for cancer and fibrosis.

Monoclonal antibodies raised against purified acetylcholine receptor from muscle and electric organ were tested for cross-reaction with surface components on chicken ciliary ganglion neurons. Indirect immunofluorescence indicated that antibodies to a determinant in the "main immunogenic region" of the receptor bind to the neurons in culture. Ultrastructural studies on 16-day embryonic ganglia, using horseradish peroxidase-conjugated monoclonal antibody, revealed that most of the conjugate labeling was associated with synaptic membrane on the neurons. A lesser amount of labeling was associated with the short processes extending from the neuronal somata in the region of preganglionic innervation. The labeling was blocked by coincubation with unlabeled antibodies of appropriate specificity and not by nonimmune serum. The pattern of labeling was clearly different from that previously found for a horseradish peroxidase conjugate of a-bungarotoxin: the toxin conjugate bound extensively to the short processes but not to synaptic membrane on the neurons. The synaptic antigen identified here by the crossreacting antibodies is a candidate for the synaptic acetylcholine receptor on chicken ciliary ganglion neurons.Chicken ciliary ganglion neurons have nicotinic acetylcholine (AcCho) receptors that mediate chemical synaptic transmission through the ganglion (1, 2). Efforts to study the regulation and distribution of such receptors on neurons have been limited by the absence of suitable probes. a-Bungarotoxin has been a very useful probe for studying nicotinic AcCho receptors in vertebrate skeletal muscle and electric organ, where the toxin binds tightly and specifically to the receptor and blocks its function (3). Ciliary ganglion neurons also have high-affinity binding sites for a-bungarotoxin, but in this case the binding sites appear to be distinct from synaptic AcCho receptors. Ultrastructural studies demonstrate that toxin binding sites on the neurons are located in the immediate vicinity of presynaptic terminals but are not present in the postsynaptic membrane (4), as would be expected for synaptic receptors. Moreover, a-bungarotoxin does not block AcCho receptor function on the neurons (5, 6), and, when the levels of AcCho sensitivity and toxin binding are compared for the neurons grown under various conditions in cell culture, no simple correlation is found between the two properties (7), as would be expected if they represented the same membrane component.An alternative approach is to use antibodies against the AcCho receptor as probes. Monoclonal antibodies (mAbs) against AcCho receptors from skeletal muscle and electric organ have been useful in studying the structure and synthesis of these receptors (8)(9)(10)(11)(12). Recent studies have shown that mAbs specific for each of the four subunits of AcCho receptors from muscle and electric organ bind to neurons in the lateral spiriform nucleus of chicken brain (13). These neurons do not bind a-bungarotoxin. Best cross-reaction was obtained with mAbs to the "...

ResultsUsing PCR, we constructed chimeric subunits in which the α7 cytoplasmic loop immediately after TM3 and before TM4 was replaced with the homologous region of the α3 or α5 sequence (Fig. 1a). The N-terminus up to TM2 regulates intersubunit assembly into receptor complexes, and α7 subunits do not coassemble with nAChR subunits, as demonstrated for endogenous subunits in CG neurons and for chimeric α7 subunits expressed in Xenopus laevis oocytes 4,5,12,13 . In particular, we have observed that coexpression of chimeric α7 subunits containing the α3 cytoplasmic loop together with wild-type α3 and β4 subunits in Xenopus oocytes results in the formation of two distinct receptor types that have the pharmacological properties of Bgt-nAChRs and nAChRs, respectively (data not shown). Thus, a difference in the distribution of chimeric α7 as compared to wild-type α7 on the infected CG neuron surface would result from the added α3 or α5 cytoplasmic loop. It cannot be explained by assembly with an endogenous nAChR subunit that can target to the synapse. Different types of neurotransmitter receptors coexist within single neurons and must be targeted to discrete synaptic regions for proper function. In chick ciliary ganglion neurons, nicotinic acetylcholine receptors (nAChRs) containing α3 and α5 subunits are concentrated in the postsynaptic membrane, whereas α-bungarotoxin receptors composed of α7 subunits are localized perisynaptically and excluded from the synapse. Using retroviral vector-mediated gene transfer in vivo, we show that the long cytoplasmic loop of α3 targets chimeric α7 subunits to the synapse and reduces endogenous nAChR surface levels, whereas the α5 loop does neither. These results show that a particular domain of one subunit targets specific receptor subtypes to the interneuronal synapse in vivo. Moreover, our findings suggest a difference in the mechanisms that govern assembly of interneuronal synapses as compared to the neuromuscular junction in vertebrates.1998 Nature America Inc.• http://neurosci.nature.com 1998 Nature America Inc.• http://neurosci.nature.com

Tissue remodeling is critical during development and wound healing. It also characterizes a number of pathologic conditions, including chronic inflammation, fibrosis and cancer. It is well appreciated that reactive stromal cells play critical roles in these settings. However, understanding of the mechanisms involved in the differentiation of reactive stromal cells and their biologic activities has been hampered by the fact that they are generated from diverse progenitors, and by their phenotypic and function heterogeneity. Furthermore, molecular markers that are expressed by all reactive stromal cells and that distinguish them from all other cell types have been lacking. Fibroblast activation protein (FAP) is a serine protease that was originally discovered as a cell surface protein expressed on astrocytomas and sarcomas. Over the last two decades, FAP has attracted increasing attention as a selective marker of carcinoma-associated fibroblasts (CAFs) and more broadly, of activated fibroblasts in tissues undergoing remodeling of their extracellular matrix (ECM) due to chronic inflammation, fibrosis or wound healing. Herein we review the evidence that FAP is indeed a robust and selective marker for reactive mesenchymal stromal cells associated with pathophysiologic tissue remodeling. We also review recent insights obtained using FAP as a tool to define the relationship between subpopulations of reactive stromal cells in various settings of tissue remodeling. Furthermore, we review recent genetic and pharmacologic data indicating that FAP and FAP-expressing cells play important roles in such conditions. Finally, we discuss the potential risks and therapeutic benefits of targeting FAP and FAP-expressing cells, as well as approaches to do so.