Transforming growth factor-α and other growth factors stimulate cell division in olfactory epithelium in vitro

The rate of cell division in olfactory epithelium (OE) is upregulated by ablation of the olfactory bulb (Carr and Farbman, 1992), or downregulated by occlusion of a naris. We used an organ culture assay of fetal rat olfactory mucosa to study regulation of the mitotic rate. Addition of any one of three members of the epidermal growth factor (EGF) family—EGF, transforming growth factor-α (TGF-α), or amphiregulin (AR)—to a serum-free culture medium resulted in a two- to threefold increase in the number of dividing OE cells. TGF-α elicited a maximal response in a dose of 100–200 pM culture medium and was 2 orders of magnitude more potent than the other EGF family members. Addition of TGF-β1, TGF-β2, insulinlike growth factor-1 or platelet-derived growth factor to the culture medium had slightly less effect than EGF or AR, in about the same molar dose range; addition of nerve growth factor had virtually no net effect on cell division. Immunohistochemistry on adult rat OE showed that basal cells, supporting cells, and acinar cells of Bowman's glands were immunoreactive with antibody to TGF-α but not with antibody to EGF. Most growth factors upregulated division of both olfactory neuron progenitors and supporting cells. The data suggest that several growth factors, most prominently TGF-α, may participate in the mitotic regulation of OE. © 1996 John Wiley & Sons, Inc.     

Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β

Cultured human melanoma cells were found to secrete TGF-β mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-β in the range from 370 to 610 pg per 10⁶ cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface-bound plasmin by the activation of plasminogen with surface-bound tissue-type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF-β activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-β present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-β1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-β produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas. © 1996 Wiley-Liss, Inc.     

Molecular dynamics simulations of epidermal growth factor and transforming growth factor-α structures in water

AMBER v. 4.1 force field in 1.5 ns NPT molecular dynamics simulations of murine epidermal growth factor (mEGF), human epidermal growth factor (hEGF), and human transforming growth factor-α (hTGF-α) structures with explicit TIP3P solvation were used to investigate differences in backbone stability, changes in secondary structure, interdomain flexibility, and weakly polar interactions. Backbone root mean square deviations of sections of each peptide show that the most stable regions in mEGF and hEGF are the A-, B-, and C-loops, whereas the most stable regions in hTGF-α are the A- and B-loops. The secondary structure in the B-loops of mEGF and hEGF differ significantly from the nuclear magnetic resonance (NMR) structures of mEGF and hEGF. The position and type of turns in the B-loop of mEGF and hEGF increase the interstrand distance of the antiparallel β-sheets thereby disrupting their structure. The interdomain flexibility of simulated hTGF-α structure is greater than in either mEGF or hEGF. The ϕ, ψ dihedrals of hTGF-α occupy two distinct populations of phase space corresponding to either a C or an α-helical conformation. This change in dihedral angle is stabilized by Phe¹⁵ with Arg⁴² and Phe¹⁷ with Arg⁴² N-π weakly polar interactions that are present only in hTGF-α but not in mEGF or hEGF. Proteins 33:396–407, 1998. © 1998 Wiley-Liss, Inc.     

Role of the transforming growth factor-β family in the expression of cranial neural crest-specific phenotypes

Cranial and trunk neural crest cells produce different derivatives in vitro. Cranial neural crest cultures produce large numbers of cells expressing fibronectin (FN) and procollagen I (PCol I) immunoreactivities, two markers expressed by mesenchymal derivatives in vivo. Trunk neural crest cultures produce relatively few FN or PCol I immunoreactive cells, but they produce greater numbers of melanocytes than do cranial cultures. Treatment of trunk neural crest cultures with transforming growth factor-β1 (TGF-β1) stimulates them to express both FN and PCol I immunoreactivities at levels comparable to those normally seen in cranial cultures and simultaneously decreases their expression of melanin. These observations raised the possibility that endogenous TGF-β is involved in specifying differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro. Consistent with this idea, we found that treatment of cranial cultures with a function-blocking TGF-β antiserum inhibits the development of FN immunoreactive cells and stimulates the development of melanocytes. Cranial and trunk neural crest cells express approximately equal levels of TGF-β mRNA. However, trunk neural crest cells are significantly less sensitive to the FN-inducing effect of TGF-β1 than are cranial neural crest cells. These results suggest that: (1) endogenous TGF-β is required for the expression of mesenchymal phenotypes by cranial neural crest cells, and (2) differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro result in part from differences in the sensitivities of these two cell populations to TGF-β. © 1995 John Wiley & Sons, Inc.     

Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin‐1α and β

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)‐β and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF‐β‐induced expression of CTGF in fibroblasts by an interleukin (IL)‐1 α‐dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL‐1α and β. Human dermal fibroblasts and NIH 3T3 cells were treated with IL‐1α or β in presence or absence of TGF‐β1. IL‐1 suppressed basal and TGF‐β‐induced CTGF mRNA and protein expression. IL‐1α and β inhibited TGF‐β‐stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3‐binding CAGA elements. Furthermore, IL‐1α and β inhibited TGF‐β‐stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF‐β activated kinase1 (TAK1) is necessary for IL‐1 inhibition of TGF‐β‐stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226–1233, 2010. Published 2010 Wiley‐Liss, Inc.     

Protein secretion by the mouse blastocyst: Stimulatory effect on secretion into the blastocoel by transforming growth factor-α

We have previously demonstrated that newly synthesized proteins are secreted into the mouse blastocoel [Dardik and Schultz (1991): Biol Reprod 45:328–333]. In the present study we examine the effect of transforming growth factor-α (TGF-α) on these proteins. We observe that TGF-α stimulates secretion of these newly synthesized proteins into the blastocoel and apical medium, which faces the zona pellucida, by about 65%. Although one-dimensional gel electrophoretic analysis does not reveal any marked differences in the patterns of newly synthesized proteins secreted into the blastocoel in response to TGF-α, zymography reveals a marked stimulation in the secretion of several gelatinases into the blastocoel and apical medium. These results suggest additional functions for TGF-α in mouse preimplantation development. © 1993 Wiley-Liss, Inc.     

Density-induced down regulation of epidermal growth factor receptors

Summary Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-β (TGF-β) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors. This work was supported by grants from the Nebraska Department of Health (89-51), the National Cancer Institute (Laboratory Research Center Support Grant, CA36727), and the American Cancer Society (Core Grant ACS SIG-16). EDITOR'S STATEMENT The paper by Rizzino et al. demonstrates that receptor number decreases as a function of cell density. This may represent a mechanism by which cell proliferation is reduced as cell density increases.     

Tumor necrosis factor-α induces substance P in sympathetic ganglia through sequential induction of interleukin-1 and leukemia inhibitory factor

The present study establishes that tumor necrosis factor-α (TNF-α) induction of sympathetic substance P (SP) requires sequential induction of both interleukin (IL-1) and leukemia inhibitory factor (LIF). TNF-α dose-dependently induces SP, an induction that is secondory to an increase in the SP precursor, preprotachykinin (PPT), mRNA. Since TNF-α conditioned medium (CM) mimics the effect of TNF-α by raising SP, actions that are not antagonized by a neutralizing TNF-α antibody, TNF-α induction of SP is mediated by a soluble intermediate or intermediates. The blockade of TNF-α action by a specific IL-1 receptor antagonist and the induction of IL-1 mRNA by TNF-α suggest that IL-1 is one of the intermediates. Moreover, because immunoprecipitation with LIF antibodies decreases SP-inducing activity of TNF-α CM, and because LIF mRNA is also induced by TNF-α, LIF is a second intermediate. Furthermore, TNF-α-induced LIF mRNA is blocked by the IL1 receptor antagonist, whereas IL-1-induced LIF mRNA is not affected by TNF-α antibodies, suggesting that TNF-α first induces IL-1, and IL-1 subsequently induces LIF. These data suggest that TNF-α induces SP in sympathetic ganglia through the sequential inductions of IL1 and LIF. © 1995 John Wiley & Sons, Inc.     

FGF2 suppresses neuronogenesis of a cell line derived from rat olfactory epithelium

Neurogenesis continues throughout adulthood in the mammalian olfactory epithelium (OE), and both neurons as well as nonneuronal cells are reconstituted following experimental injury. Underlying the capacity of the OE to replenish its mature elements is a population of progenitor basal cells. Although the precise lineage relationships among progenitor and mature cell types are incompletely understood, the population of globose basal cells (GBCs) contains immediate precursors to neurons as well as amplifying progenitors, and retroviral lineage analyses suggest that multipotential GBCs are activated following direct injury to the OE. To assess the controls on the process of epithelial regeneration, we have characterized a cell line derived from rat OE and studied the effects of serum and tissue extracts, fibroblast growth factor-2 (FGF2) and transforming growth factor-α (TGFα) on the cells. Using a panel of cell type-specific markers whose patterns of labeling in the OE are well defined, including recently developed markers for GBCs, we characterized the phenotype of the cell line under differing culture conditions. In complete medium, which contains serum and tissue extracts, the cell line displayed characteristics of GBCs that are prominent during regeneration. Serum and extract withdrawal induced the cells to differentiate into neurons. In contrast, FGF2 prevented neuronal differentiation and maintained a GBC phenotype. TGFα had a mitogenic or differentiative effect that was context dependent. Finally, we demonstrate here that FGF2 is contained in mature olfactory neurons and sustentacular cells in vivo, suggesting a physiologic role for this growth factor in OE cell regulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 411–428, 1997     

Transforming growth factor-β1 inhibits regeneration of renal proximal tubular cells after oxidant exposure

Transforming growth factor-β (TGF-β), a regulatory cytokine expressed in the kidney, plays a role in nephrogenic repair. This study utilized a chemical model of renal proximal tubule cellular injury and regeneration to investigate the effects of TGF-β₁ on regeneration. Confluent monolayers of rabbit renal proximal tubular cells (RPTC) in primary culture exposed to the oxidant t-butylhydroperoxide (800 μM TBHP) for 1.5 hours were 24% confluent after 24 hours. Confluency increased to 50% 4 days after TBHP exposure. Recovery of monolayer confluency was associated with increased monolayer protein but not with DNA content. Daily treatment of injured monolayers with TGF-β₁ inhibited the recovery of monolayer confluency and inhibited recovery of protein content in a concentration-dependent manner (0.02–1 ng/mL). DNA content of injured monolayers was not altered by TGF-β₁. A single treatment of injured monolayers with 0.2 ng/mL (8 pM) TGF-β₁ inhibited recovery of monolayer confluency and protein content without altering monolayer DNA content. These data show that a single 24 hour exposure to a low concentration (8 pM) of TGF-β₁ inhibits regeneration of renal proximal tubule cell monolayers following oxidative injury by inhibiting, in part, cellular migration/spreading. © 1996 John Wiley & Sons, Inc.     

CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-β1

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-β1 and proinflammatory interleukin (IL)-1β are widely associated with fibrotic progression. TGF-β1 induces myofibroblast differentiation, while IL-1β induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-β1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-β1- and IL-1β-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-β1-induced fibroblast/myofibroblast differentiation and IL-1β-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell–cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.     

Early defect of transforming growth factor β1 formation in Huntington’s disease

A defective expression or activity of neurotrophic factors, such as brain‐ and glial‐derived neurotrophic factors, contributes to neuronal damage in Huntington’s disease (HD). Here, we focused on transforming growth factor‐β (TGF‐β₁), a pleiotropic cytokine with an established role in mechanisms of neuroprotection. Asymptomatic HD patients showed a reduction in TGF‐β₁ levels in the peripheral blood, which was related to trinucleotide mutation length and glucose hypometabolism in the caudate nucleus. Immunohistochemical analysis in post‐mortem brain tissues showed that TGF‐β₁ was reduced in cortical neurons of asymptomatic and symptomatic HD patients. Both YAC128 and R6/2 HD mutant mice showed a reduced expression of TGF‐β₁ in the cerebral cortex, localized in neurons, but not in astrocytes. We examined the pharmacological regulation of TGF‐β₁ formation in asymptomatic R6/2 mice, where blood TGF‐β₁ levels were also reduced. In these R6/2 mice, both the mGlu2/3 metabotropic glutamate receptor agonist, LY379268, and riluzole failed to increase TGF‐β₁ formation in the cerebral cortex and corpus striatum, suggesting that a defect in the regulation of TGF‐β₁ production is associated with HD. Accordingly, reduced TGF‐β₁ mRNA and protein levels were found in cultured astrocytes transfected with mutated exon 1 of the human huntingtin gene, and in striatal knock‐in cell lines expressing full‐length huntingtin with an expanded glutamine repeat. Taken together, our data suggest that serum TGF‐β₁ levels are potential biomarkers of HD development during the asymptomatic phase of the disease, and raise the possibility that strategies aimed at rescuing TGF‐β₁ levels in the brain may influence the progression of HD.     

The circular dichroism of tumor necrosis factor-α: Measurement into the vacuum UV and analysis for secondary structure

The circular dichroism (CD) spectrum of tumor necrosis factor-α has been measured into the vacuum UV to 168 nm. Analysis of the CD for secondary structure is in good agreement with X-ray diffraction results, but the analysis is somewhat unstable. Adding the CD of this protein together with its X-ray determined secondary structure to the basis set should improve subsequent analyses of CD spectra for other all-β proteins.     

Modulation of type alpha transforming growth factor receptors by a phorbol ester tumor promoter

Epidermal growth factor (EGF) and an EGF-like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37 degrees C induces a complete loss of high-affinity (Kd = 35-50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half-maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF/eTGF receptors. The EGF/eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. We conclude that the tumor-promoting phorbol diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type alpha transforming growth factors, eTGF and EGF.     

Effect of tumor necrosis factor-α on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases

Tumor necrosis factor-α (TNF-α) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-α has been shown to mediate the insulin resistance associated with animal models of obesity and noninsulin-dependent diabetes mellitus. In order to determine whether the effects of TNF-α might involve alterations in the expression of specific protein-tyrosine phosphatases (PTPases) that have been implicated in the regulation of growth factor receptor signalling, KRC-7 rat hepatoma cells were treated with TNF-α, and changes in overall tissue PTPase activity and the abundance of three major hepatic PTPases (LAR, PTP1B, and SH-PTP2) were measured in addition to effects of TNF-α on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-α caused a dose-dependent decrease in insulin-stimulated IRS-1 phosphorylation and EGF-stimulated receptor autophosphorylation to 47–50% of control. Overall PTPase activity in the cytosol fraction did not change with TNF-α treatment, and PTPase activity in the particulate fraction was decreased by 55–66%, demonstrating that increases in total cellular PTPase activity did not account for the observed alterations in receptor signalling. However, immunoblot analysis showed that TNF-α treatment resulted in a 2.5-fold increase in the abundance of SH-PTP2, a 49% decrease in the transmembrane PTPase LAR, and no evident change in the expression of PTP1B. These data suggest that at least part of the TNF-α effect on pathways of reversible tyrosine phosphorylation may be exerted through the dynamic modulation of the expression of specific PTPases. Since SH-PTP2 has been shown to interact directly with both the EGF receptor and IRS-1, increased abundance of this PTPase may mediate the TNF-α effect to inhibit signalling through these proteins. Furthermore, decreased abundance of the LAR PTPase, which has been implicated in the regulation of insulin receptor phosphorylation, may account for the less marked effect of TNF-α on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited. J. Cell. Biochem. 64:117–127. © 1997 Wiley-Liss, Inc.