Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII

We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII-Arg100, the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII-Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.     

Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C

Thrombomodulin is a cofactor protein on vascular endothelial cells that inhibits the procoagulant functions of thrombin and enhances thrombin-catalyzed activation of anticoagulant protein C. Thrombomodulin also accelerates the proteolytic activation of a plasma procarboxypeptidase referred to as thrombin-activable fibrinolysis inhibitor (TAFI). In this study, we describe structures on recombinant membrane-bound thrombomodulin that are required for human TAFI activation. Deletion of the N-terminal lectin-like domain and epidermal growth factor (EGF)-like domains 1 and 2 had no effect on TAFI or protein C activation, whereas deletions including EGF-like domain 3 selectively abolished thrombomodulin cofactor activity for TAFI activation. Provided that thrombomodulin EGF-like domain 3 was present, TAFI competitively inhibited protein C activation catalyzed by the thrombin-thrombomodulin complex. A thrombomodulin construct lacking EGF-like domain 3 functioned normally as a cofactor for protein C activation but was insensitive to inhibition by TAFI. Thus, the anticoagulant and antifibrinolytic cofactor activities of thrombomodulin have distinct structural requirements: protein C binding to the thrombin-thrombomodulin complex requires EGF-like domain 4, whereas TAFI binding also requires EGF-like domain 3.     

The membrane-bound form of heparin-binding epidermal growth factor-like growth factor promotes survival of cultured renal epithelial cells

To understand whether expression of membrane-anchored heparin binding epidermal growth factor (proHB-EGF) is involved in renal epithelial cell survival, rat membrane-bound HB-EGF precursor was stably transfected into a renal epithelial cell line, NRK 52E cells (NRKproHB-EGF). When exposed to 10% fetal calf serum (FCS), there were no differences in growth rates among wild-type (WT), vector-transfected (NRKvector), and NRKproHB-EGF. However, when cells were grown in the presence of 1% FCS, the growth rate of NRKproHB-EGF was 65% faster. When confluent cell monolayers were exposed to H2O2 or etoposide, WT or NRKvector exhibited significant apoptotic bodies and DNA laddering; in contrast, NRKproHB-EGF were resistant to both stimuli, as indicated by increased cell viability and marked decrease of apoptotic bodies and DNA laddering. When plated at high density onto plastic dishes without FCS, WT and NRKvector formed few attachments, did not proliferate, and underwent apoptosis. By day 3, no cells survived. Addition of exogenous recombinant HB-EGF (10(-8) M) to WT or NRKvector increased cell survival by <10% and incubation with conditioned media of NRKproHB-EGF had no effect. In contrast, NRKproHB-EGF attached and formed epithelial colonies, although they did not proliferate. After 3 days, cell viability was 84% of the initial cell number plated, and no evidence of apoptosis was present. When plated in 10% FCS, NRKproHB-EGF attachment to plastic substratum at 1, 2, and 3 h was 250% greater than that of WT or NRKvector. Addition of exogenous recombinant human HB-EGF to WT or NRKvector increased attachment by <50%. When grown on poly(2-hydroxyethyl methacrylate) or in the presence of the integrin receptor-blocking peptide GRGDTP, neither WT nor NRKvector attached to the substratum or formed cell-cell attachments. Compared with WT or NRKvector, NRKproHB-EGF exhibited 300% greater cell viability on either poly(2-hydroxyethyl methacrylate)-coated dishes or in the presence of GRGDTP and formed cell clusters. When plated at low density (1 x 10(3) cells/1.5-cm dish) or at high density in the presence of an anti-HB-EGF blocking antibody, NRKproHB-EGF failed to form epithelial colonies. Addition of formalin fixed NRKproHB-EGF promoted EGF receptor tyrosine phosphorylation in quiescent A431 cells and stimulated DNA synthesis and prevented H2O2-induced apoptosis in renal epithelial cells. These results indicate that membrane-bound HB-EGF promotes renal epithelial cell survival, possibly by promoting cell-matrix and cell-cell interactions. The failure of either conditioned media or exogenous HB-EGF to reproduce these findings suggests that juxtacrine or tightly coupled paracrine interactions underlie this cytoprotection.     

Heparin-binding epidermal growth factor-like growth factor in experimental models of membranous and minimal change nephropathy

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently described member of the epidermal growth factor (EGF) family. It binds to heparan sulfate proteoglycans via a cationic domain and is a potent mitogen for epithelial cells, fibroblasts and vascular smooth muscle cells. In the present study we have attempted to identify changes in quantity and distribution of HB-EGF in two models of acute glomerular epithelial cell injury, using Western blotting, immunohistochemistry and in situ hybridization. Prior to disease induction, Western blots showed some expression of HB-EGF protein within glomeruli. Within the first three days in the acute puromycin aminonucleoside (PAN) and passive Heymann nephritis (PHN) models, immunohistochemistry and in situ hybridization demonstrated an up-regulation of HB-EGF mRNA and protein in glomerular epithelial cells (GEC). In both cases, increased protein and mRNA was found prior to the onset of proteinuria and continued until day 21 post-induction, the last time point studied. Early in the course of the models, HB-EGF was localized to the cytoplasm of glomerular epithelial cells. At day 21, however, HB-EGF protein was distributed in a nodular pattern within GEC and along the glomerular basement membrane (GBM) in both models, suggesting that the secreted form might bind to the membrane. The increase in HB-EGF protein within glomeruli was confirmed by Western blots of glomerular membrane protein which, however, demonstrated a single 29 kDa species, consistent with the transmembrane form. These data are not consistent with binding of the secreted form of HB-EGF to the GBM. The transmembrane form of HB-EGF is able to signal in a juxtracrine fashion, so increased expression of HB-EGF mRNA and protein by GEC might contribute to the genesis of proteinuria through the initiation of abortive GEC mitogenesis.     

Immunohistochemical localization of heparin-binding epidermal growth factor-like growth factor in normal skin and skin cancers

Heparin-binding epidermal growth factor (EGF)-like growth factor is a 22-kDa glycoprotein that was originally identified as a secreted product of cultured human macrophages. Although the growth factor mRNA has been identified in various cells and tissues, the tissue distribution of the protein itself has rarely been demonstrated. In this study, the EGF-like growth factor was detected immunohistochemically in a variety of human skin samples by indirect immunofluorescence using a polyclonal rabbit antiserum raised against residues 26-41 of mature heparin-binding EGF. The keratinocytes of a variety of epithelium-derived structures demonstrated reproducible, specific staining for the EGF. In normal tissues, this staining was prominent in the basal cells of the epidermis and in the epithelial cells lining epidermal appendages such as hair follicles, sebaceous sweat glands and eccrine sweat glands. In addition, specific staining was detected in skin cancers derived from the basal epithelial cell layer, including basal and squamous cell carcinomas of the skin, with no staining detected in melanoma specimens. Immunoreactive heparin-binding EGF was characteristically associated with the surface of cells. With minor exceptions, the immunoreactive sites are identical to the known EGF receptor distribution in the skin, and suggest that keratinocyte-derived heparin-binding EGF may act in concert with other EGF family members in processes such as skin morphogenesis and wound repair, as well as in the development of skin cancers.     

RGS3 inhibits G protein-mediated signaling via translocation to the membrane and binding to Galpha11

In the present study, we investigated the function and the mechanism of action of RGS3, a member of a family of proteins called regulators of G protein signaling (RGS). Polyclonal antibodies against RGS3 were produced and characterized. An 80-kDa protein was identified as RGS3 by immunoprecipitation and immunoblotting with anti-RGS3 antibodies in a human mesangial cell line (HMC) stably transfected with RGS3 cDNA. Coimmunoprecipitation experiments in RGS3-overexpressing cell lysates revealed that RGS3 bound to aluminum fluoride-activated Galpha11 and to a lesser extent to Galphai3 and that this binding was mediated by the RGS domain of RGS3. A role of RGS3 in postreceptor signaling was demonstrated by decreased calcium responses and mitogen-activated protein (MAP) kinase activity induced by endothelin-1 in HMC stably overexpressing RGS3. Moreover, depletion of endogenous RGS3 by transfection of antisense RGS3 cDNA in NIH 3T3 cells resulted in enhanced MAP kinase activation induced by endothelin-1. The study of intracellular distribution of RGS3 indicated its unique cytosolic localization. Activation of G proteins by AlF4-, NaF, or endothelin-1 resulted in redistribution of RGS3 from cytosol to the plasma membrane as determined by Western blotting of the cytosolic and particulate fractions with RGS3 antiserum as well as by immunofluorescence microscopy. Agonist-induced translocation of RGS3 occurred by a dual mechanism involving both C-terminal (RGS domain) and N-terminal regions of RGS3. Thus, coexpression of RGS3 with a constitutively active mutant of Galpha11 (Galpha11-QL) resulted in the binding of RGS3, but not of its N-terminal fragment, to the membrane fraction and in its interaction with Galpha11-QL in vitro without any stimuli. However, both full-length RGS3 and its N-terminal domain translocated to the plasma membrane upon stimulation of intact cells with endothelin-1 as assayed by immunofluorescence microscopy. The effect of endothelin-1 was also mimicked by calcium ionophore A23187, suggesting the importance of Ca2+ in the mechanism of redistribution of RGS3. These data indicate that RGS3 inhibits G protein-coupled receptor signaling by a complex mechanism involving its translocation to the membrane in addition to its established function as a GTPase-activating protein.     

High concentration of glucose increases mitogenic responsiveness to heparin-binding epidermal growth factor-like growth factor in rat vascular smooth muscle cells

The effect of a high extracellular glucose concentration on the mitogenic response of rat vascular smooth muscle cells (SMCs) to heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated. The mitogenic effect of HB-EGF was significantly greater in SMCs cultured in high glucose (25 mmol/L) than in cells cultured in low glucose (5.5 mmol/L) or at high osmolarity (5.5 mmol/L glucose plus 19.5 mmol/L mannitol). The mitogenic effect of epidermal growth factor (EGF), which shares the EGF receptor with HB-EGF, was not affected by glucose concentration. The mitogenic effect of HB-EGF was greater when incubated with heparan sulfate (HS) isolated from SMCs cultured in high glucose than with HS from cells cultured in low glucose. HS synthesized by cells in high glucose was of smaller molecular size and less sulfated than HS synthesized by cells in low glucose. The abundance of mRNA encoding HS-N-deacetylase/N-sulfotransferase (HS-NdAc/NST), a regulatory enzyme in the biosynthesis of HS, was decreased by high glucose in a protein kinase C-independent manner. These observations suggest that the enhanced mitogenic response to HB-EGF in SMCs cultured in high glucose may be attributable to changes in cell-associated HS. Downregulation of HS-NdAc/NST gene expression by high glucose may be related to the altered HS biosynthesis.     

Requirement for c-Src catalytic activity and the SH3 domain in platelet-derived growth factor BB and epidermal growth factor mitogenic signaling

The Src family protein-tyrosine kinases are required for mitogenic signaling from the platelet-derived growth factor (PDGF), colony stimulating factor-1, and epidermal growth factor (EGF) receptor protein-tyrosine kinases (RPTK) (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700; Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtneidge, S. A.(1995) Mol. Cell. Biol. 15, 1102-1109). In NIH3T3 fibroblasts, c-Src, Fyn, and c-Yes associate with the activated PDGF receptor, are substrates for receptor phosphorylation, and are themselves activated. Src family catalytic function is required for RPTK mitogenic signaling as evidenced by the SH2-dependent dominant negative phenotype exhibited by kinase-inactive Src and Fyn mutants (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700). Here, we have generated clonal Src- murine fibroblast cell lines overexpressing various murine c-Src mutants and studied the effect of these mutant Src proteins on PDGF- and EGF-induced mitogenesis. Two c-Src SH3 domain mutants, Y133F and Y138F, each inhibited PDGF BB- and EGF-induced DNA synthesis in quiescent cells. This demonstrates an involvement of the Src SH3 domain in PDGFbeta and EGF receptor mitogenic signaling. Since both Tyr-133 and Tyr-138 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling. The dominant negative effect of either single mutant on PDGF receptor signaling was reversed by a second SH2-inactivating mutation. We conclude that the c-Src SH3 domain function requires the SH2 domain in the case of the PDGF receptor, presumably because binding of c-Src to the receptor via its SH2 domain is a prerequisite for the SH3 domain function. In contrast, SH2 function is apparently not essential for the SH3 function in EGF receptor signaling.     

A membrane setting for the sorting motifs present in the adenovirus E3-13.7 protein which down-regulates the epidermal growth factor receptor

The adenovirus E3-13.7 protein interferes with endosomal protein sorting to down-regulate the epidermal growth factor receptor and related tyrosine kinase receptors. The cytoplasmic C terminus of this protein contains three protein sorting motifs which are related to the function of E3-13.7. In this study, the structure of a 23-residue polypeptide corresponding to this domain was examined using solution NMR and CD spectroscopic methods. The peptide was observed to exist in a mostly random structural state in aqueous solution but underwent high affinity association with dodecylphosphocholine micelles, where it adopted an ordered structure. The affinity of this peptide for the micellar surface and the structure of the bound peptide were independent of pH variation, surface charge, or attachment of a myristoyl anchor to the N-terminal. Studies with phospholipid vesicles suggested that the micellar structural results can be extrapolated to a true lipid bilayer. On the micellar surface all three sorting motifs are closely associated with the water/apolar interface: 72-YLRH and 87-LL lie within interfacial amphipathic helices, while 76-HPQY is non-helical and dimples just above the surface. These results contribute to the development of an understanding of the basis for specificity in recognition of sorting motifs by components of the cellular protein trafficking machinery.     

Menadione (vitamin K3) enhances the mitogenic signal of epidermal growth factor via extracellular signal-regulated kinases

The effects of vitamin K3 (VK3) on DNA synthesis, cell proliferation and mitogen-activated protein kinase pathway were investigated in G0-arrested NIH 3T3 fibroblasts. VK3 (5 microM) alone stimulates DNA synthesis by 40% and moderately increases the mitogenic effects of EGF, which is preceded by a rapid phosphorylation of the extracellular signal-regulated kinases (ERKs). At 20 microM, VK3 had an antiproliferative effect. VK3 alone (5 and 50 microM) or in concert with EGF increases the activity of ERK2 (by 2.5 and 5 fold, respectively). Our studies demonstrate that the activation of ERKs by VK3 alone, or VK3 plus EGF can promote either stimulatory or inhibitory effects on the mitogenic signal.     

Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain

The bovine papillomavirus E5 transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. All chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that E5 activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that E5 formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for E5 activation, both receptors are activated directly by formation of an E5-containing complex.     

Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity

The membrane anchored form of human heparin-binding epidermal growth factor-like growth factor (HB-EGF) acts as the diphtheria toxin (DT) receptor. Transfection of human HB-EGF cDNA into mouse LC cells, L cells stably expressing DRAP27, conferred sensitivity to DT, but transfection of mouse HB-EGF cDNA did not. To define the essential regions of HB-EGF that serve as the functional DT receptor, we examined the sensitivity to DT and DT binding of cells expressing several human/mouse HB-EGF chimeras. It was found that DT binds to the EGF-like domain of the human HB-EGF. However, mouse HB-EGF does not serve as a functional DT receptor due to non-conserved amino acid substitutions in this domain. In addition, CRM197, a non-toxic mutant of DT, inhibited strongly the mitogenic activity of the secreted form of human HB-EGF, but not of mouse HB-EGF and other EGF receptor-binding growth factors. These results confirmed further that DT interacts with the EGF-like domain of HB-EGF and that this interaction is specific for human HB-EGF.     

Dehydroepiandrosterone inhibits the growth of DMBA-induced rat mammary carcinoma via the androgen receptor

Dehydroepiandrosterone (DHEA) exerts opposite effects on the growth of mammary carcinoma. A stimulatory effect is observed in absence of estrogens, due to interaction of DHEA metabolite(s) with the estrogen receptor (ER); by contrast, in presence of estrogens DHEA inhibits tumor growth. The mechanism underlying the latter DHEA effect, which might be related to activation of the androgen receptor (AR), is poorly understood. Focusing on this point, we measured over a 20 days period the areas of DMBA-induced mammary tumors in rats given DHEA and/or the anti-androgen flutamide (FLU). Results show that DHEA inhibitory effect on the growth of mammary carcinoma is no longer observed when the ARs are blocked by FLU. Data are consistent with an involvement of ARs in the inhibitory effect of DHEA.     

Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells

Chondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine growth factor-inducible immediate-early gene, cyr61. The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. In this communication, we demonstrate that the Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis that contributes to the development of the mammalian embryonic skeleton.     

T3 receptor suppression of Sp1-dependent transcription from the epidermal growth factor receptor promoter via overlapping DNA-binding sites

Expression of the human epidermal growth factor receptor (EGFR) gene is inhibited by ligand-activated thyroid hormone receptor (T3R). Binding sites for Sp1 and for the T3R.retinoid X receptor (RXR) complex overlap in a functional core of the EGFR promoter. Sp1 inhibited binding of the T3R complex to this 36-base pair (bp) EGFR element in vitro but did not affect binding of the T3R complex to a positive thyroid hormone response element (TRE). In Drosophila SL2 cells, which lack Sp1 and T3R, function of the EGFR promoter was strongly dependent on Sp1. Sp1-dependent promoter function was inhibited by ligand-activated T3R but not by mutant T3R defective in DNA or T3 binding. RXR increased the extent of inhibition. Sp1 enhanced activity of the 36-bp element placed 5' to a minimal TATA promoter and this enhancement was also repressed by T3R. Mutations in the 36-bp element were unable to separate Sp1 and T3R functions. However, addition of a second half-site 5' to the existing site in an inverted repeat configuration created a positive TRE. In the absence of ligand, T3R inhibited Sp1 stimulation from this altered element; addition of T3 reversed the inhibition. When a dimeric TRE is separated from Sp1-binding sites strong synergism was observed. The nature and location of the TRE thus strongly influence biological responses. A TRE site in the EGFR promoter that overlaps an Sp1-binding site inhibits Sp1 function but is unable to direct positive function.     

A novel ligand for an SH3 domain of the adaptor protein Nck bears an SH2 domain and nuclear signaling motifs

Nck is a small protein composed of Src homology regions (SH) 2 and 3, paralleling the adaptors c-Crk and Grb2/Ash, but its function remains enigmatic. To clarify Nck signaling, a human brain cDNA library was searched for targets of the SH3 moiety of Nck. A novel molecule detected therefrom (referred to as Nck-, Ash- and phospholipase Cgamma-binding protein 4) contained proline-rich sequences and, through the function of one of them, interacted with the middle SH3 domain of Nck. A NAP4 fusion peptide exhibited an affinity for Nck, Ash and phospholipase Cgamma in whole cell lysates. NAP4 also had an SH2 domain, which could bind to activated EGF receptor. These intermolecular interactions imply the intricacy of Nck-mediated signaling around the receptor protein-tyrosine kinases. In addition, NAP4 bore a putative nuclear localization signal and a Q-run/P-run composite, both characteristic of nuclear proteins, and might therefore relate to the presence of Nck in the cellular nucleus.     

Modulated expression of the epidermal growth factor-like homeotic protein dlk influences stromal-cell-pre-B-cell interactions, stromal cell adipogenesis, and pre-B-cell interleukin-7 requirements

A close relationship exists between adipocyte differentiation of stromal cells and their capacity to support hematopoiesis. The molecular basis for this is unknown. We have studied whether dlk, an epidermal growth factor-like molecule that intervenes in adipogenesis and fetal liver hematopoiesis, affects both stromal cell adipogenesis and B-cell lymphopoiesis in an established pre-B-cell culture system. Pre-B-cell cultures require both soluble interleukin-7 (IL-7) and interactions with stromal cells to promote cell growth and prevent B-cell maturation or apoptosis. We found that BALB/c 3T3 fibroblasts express dlk and function as stromal cells. Transfection of these cells with antisense dlk decreased dlk expression and increased insulin-induced adipocytic differentiation. When antisense transfectants were used as stroma, IL-7 was no longer required to support the growth of pre-B cells and prevent maturation or apoptosis. Antisense dlk transfectants of S10 stromal cells also promoted pre-B-cell growth in the absence of IL-7. These results show that modulation of dlk on stromal cells can influence their adipogenesis and the IL-7 requirements of the pre-B cells growing in contact with them. These results indicate that dlk influences differentiation signals directed both to the stromal cells and to the lymphocyte precursors, suggesting that dlk may play an important role in the bone marrow hematopoietic environment.