Low Concentrations Of Epidermal Growth Factor Research Articles

Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro

In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acid-supplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor β (FGF-β) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-β concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.

Effect of seminal plasma infusion into the vagina on the normalization of endometrial epidermal growth factor concentrations and fertility in repeat breeder dairy cows

Epidermal growth factor (EGF) concentrations in the uterus show two peaks on days 2–4 and 13–14 during the estrous cycle in fertile cows. Loss of the two peaks has been linked to reduced fertility in repeat breeder cows. This study aimed to examine the effect of seminal plasma (SP) on normalizing endometrial EGF concentrations and restoring fertility in repeat breeder cows with low EGF concentrations on day 3. In study 1, we examined the effect of the deposition sites (the vagina and uterus) of SP on the endometrial EGF concentrations in repeat breeder cows. SP infusion into the vagina, but not uterus, on the first day of the estrus cycle (day 0) normalized the endometrial EGF concentrations (≥ 4.7 ng/g tissue weight) on day 3. In study 2, the effect of SP volume (0.5 and 10 ml of SP and 0.5 ml of SP diluted to 10 ml) on EGF concentrations was examined. All groups with SP infusion had increased EGF concentrations on day 3, and cows with 10 ml of SP and 0.5 ml of SP diluted to 10 ml showed the highest levels of EGF concentrations. In study 3, we examined the effect of SP infusion on fertility. SP infusion normalized two peaks of endometrial EGF concentrations in about 60% of repeat breeder cows and produced more pregnancies than the controls (44.4 vs. 19.4%). Therefore, we concluded that SP may contain an activity to normalize the EGF profile and restore fertility in repeat breeder cows with altered EGF profiles.

The positional identity of iPSC-derived neural progenitor cells along the anterior-posterior axis is controlled in a dosage-dependent manner by bFGF and EGF

Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens, including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). However, these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis.Here, we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations of bFGF and EGF. We observed that low concentrations of bFGF and EGF in the culturing system were able to induce forebrain identity of the neural rosettes and promote subsequent cortical neuronal differentiation. On the contrary, high concentrations of these mitogens stimulate a mid-hindbrain fate of the neural rosettes, resulting in subsequent cholinergic neuron differentiation. Thus, our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells.

Salivary Concentration of Epidermal Growth Factor in Adults with Reflux Laryngitis before and after Treatment

The diagnosis of laryngopharyngeal reflux (LPR) is controversial. There is no correlation between the number of reflux episodes and the severity of the inflammatory response at the esophagus or the laryngopharyngeal segment. Some authors have suggested that decreased salivary epidermal growth factor (EGF) concentrations in patients with gastroesophageal reflux disease and LPR point to a breakdown in the local defenses. Our objective was to establish whether treatment of the disease influences low salivary EGF concentrations. The spontaneous whole saliva of 20 adults with LPR was sampled at a tertiary teaching hospital before and after a 16-week course of full-dose proton pump inhibitor and compared to that of 12 healthy controls. Salivary EGF concentrations were established with a commercially available enzyme-linked immunosorbent assay kit. Although the mean salivary EGF concentrations were higher before treatment than after treatment and control of the disease (25,083 versus 19,359 pg/mL), this difference was not statistically significant (p = 0.065). The mean salivary EGF concentration of healthy control subjects was significantly higher (54,509 pg/mL; p < 0.0001). Both before and after treatment, patients with reflux laryngitis present lower salivary EGF concentrations than healthy control subjects, suggesting a primary deficit in their protective mechanisms.

Effects of Epidermal Growth Factor and Basic Fibroblast Growth Factor on the Proliferation and Osteogenic and Neural Differentiation of Adipose-Derived Stem Cells

Stem cells used for clinical tissue regeneration therapy should have the capacity of self-renewal, high proliferation, and differentiation and be able to be transplanted in large numbers. Although high concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) may induce the differentiation of stem cells, these factors have been widely used to enhance the propagation of stem cells, including adipose-derived mesenchymal stem cells (ASCs). However, the effects of low concentrations of EGF and bFGF on stem cells need to be evaluated carefully. This study illustrates that low concentrations of EGF (5 ng/mL) and bFGF (10 ng/mL) increase the proliferative ability of ASCs and induce the typical spindle-shaped cell morphology. EGF and bFGF added to medium promoted neural lineage differentiation and impaired the mesodermal differentiation ability of ASCs. This study demonstrates that even low concentrations of EGF and bFGF may limit the differentiation ability of stem cells during stem cell expansion in vitro. EGF and bFGF supplementation should be carefully considered in stem cells for clinical applications.

Epidermal growth factor modulates voltage sensitivity of slow sodium channels

The objective of this study was to determine the mechanisms underlying excitation of the nociceptive neuronal membrane under the influence of growth factors. Each ionic channel or its gate structure is known to have a specific function during the primary sensory encoding. For example, adaptation is a result of activation of slow sodium channels or it is caused by complicated behavior of the activation gate structure of sodium channels (1, 2). Receptormediated modu� lation of the activation gate structure of slow sodium channels also reduces the frequency of recurrent responses of the nociceptive neuronal membrane (3). This antinociceptive effect may be caused by the epi� dermal growth factor (EGF). Our study demonstrates that precisely this growth factor modulates the voltage sensitivity of slow tetrodotoxininsensitive sodium channels (Na v 1.8), which are responsible for encoding the pain signal (4, 5). Growth factors are polypeptides constituting a group of trophic regulatory substances. EGF is the main polypeptide stimulating growth of endometrium cells (6). EGF polypeptide with a molecular weight of about 6 kDa is composed of 53 amino acid residues and has an effect of a strong mitogen on various cells of the endo�, ecto�, and mesodermal origin. Low EGF concentrations (above 2 ng/mL) were determined in blood, cerebrospinal fluid, milk, saliva, gastric and pancreatic juices (7).

Alteration of the Endometrial EGF Profile as a Potential Mechanism Connecting the Alterations in the Ovarian Steroid Hormone Profile to Embryonic Loss in Repeat Breeders and High-producing Cows

Poor reproductive efficiency is a worldwide problem that has affected the dairy industry during the last several decades. In an attempt to explain the changes in reproductive physiology caused by high milk production, a model of elevated steroid metabolism in lactating dairy cows has been proposed. A slow increase in levels and low peak levels of estradiol (E2) and progesterone (P4) characterize endocrine changes in high producing cows. Similar changes have been reported in the repeat breeder cows. The abnormal changes in E2 and P4 concentrations of these cows may cause an improper uterine environment due to disturbed expression of growth factors and cytokines in the endometrium. This review focuses on the alteration in epidermal growth factor (EGF) profile in the endometrium during the estrous cycle. The normal cow has two peaks of EGF concentrations on days 2–4 and 13–14. Low concentrations of EGF on these days distinguished both high-producing and repeat breeder cows from normal cows. Alteration of the EGF profile could be found in 70 and 40% of the repeat breeder and high-producing cows, respectively. Treatment with a high dose of estradiol benzoate and an intravaginal progesterone-releasing device restored the normal EGF profile in about 70% of the affected cows. The cows having a normal EGF profile after treatment showed a higher pregnancy rate than the cows with the altered profile. Further studies to understand the etiology of the alteration in the EGF profile are needed to develop another treatment option and preventive management for this problem.

Deletion of interferon-γ delays onset and severity of dacryoadenitis in CD25KO mice

IntroductionTo investigate the role of interferon-gamma (IFN-γ) in the onset and severity of dacryoadenitis in the CD25 knockout (KO) mouse model of Sjögren Syndrome.MethodsCD25/IFN-γ double KO (γDKO) mice were created by crossbreeding CD25KO and IFN-γKO mice. Mice were used at 8, 12, and 16 weeks. Lacrimal gland (LG) infiltrating lymphocytes were characterized with flow cytometry. Tear epidermal growth factor (EGF) concentration was measured with enzyme-linked immunosorbent assay (ELISA). Quantitative polymerase chain reaction (PCR) evaluated T-cell-related cytokines in LGs. Serum autoantibodies against M3R in LG lysates were detected with Western blot.ResultsγDKO LG showed lower lymphocytic infiltration at 8 weeks than in the CD25KO parental strain (˜20% versus ˜60%, respectively), which increased to CD25KO levels at 16 weeks. Flow-cytometry analysis showed an increase in CD4+ and CD8+ T cells with aging in γDKO LG, similar to that in CD25KO. γDKO had lower levels of interleukin (IL)-17A, transforming growth-factor (TGF)-β1, IL-21, and CCL20, and higher IL-1β and IL-13 mRNA transcripts in the LG than in the parental CD25KO strain. Autoantibodies to M3R were observed in both strains and significantly increased with aging in both strains. CD25KO mice had very low tear EGF concentrations at all ages, whereas the ear EGF concentration in γDKO mice significantly decreased with aging and inversely correlated with the presence of M3R autoantibodies and the degree of LG CD4 and CD8+ T-cell infiltration.ConclusionsThe deletion of IFN-γ in the CD25KO mice strain delays glandular destruction and preserves glandular function. M3R autoantibodies increased with aging in both the γDKO and the CD25KO strains. The decrease in LG function in γDKO correlated with the degree of T-cell infiltration and the presence of M3R autoantibodies.

Acute ligand-independent Src activation mimics low EGF-induced EGFR surface signalling and redistribution into recycling endosomes

Src, a non-receptor tyrosine kinase, is a key signal transduction partner of epidermal growth factor (EGF) receptor (EGFR). In human breast cancer, EGFR and Src are frequently over-expressed and/or over-activated. Although reciprocal activation is documented, mechanisms underlying Src:EGFR interactions are incompletely understood. We here exploited ts/v-Src thermo-activation in MDCK monolayers to test whether acute Src activation impacts on signalling and trafficking of non-liganded EGFR. We found that thermo-activation caused rapid Src recruitment to the plasma membrane, concomitant association with EGFR, and its phosphorylation at Y845 and Y1173 predominantly at the cell surface. Like low EGF concentrations, activated Src (i) decreased EGF surface binding without affecting the total EGFR pool; (ii) triggered EGFR endocytosis via clathrin-coated vesicles; (iii) and led to its sequestration in perinuclear/recycling endosomes with avoidance of multivesicular bodies and lysosomal degradation. Combined Src activation and EGF were synergistic for EGFR-Y845 and -Y1173 phosphorylation at some endosomes. We conclude that acute effects of Src in MDCK cells may mimic those of low EGF on EGFR activation and redistribution. Src:EGFR interactions may be sufficient to trigger EGFR activation and might contribute to its local signalling, without requiring either soluble extracellular signal or receptor over-expression.

Epidermal Growth Factor Stimulates Human Trophoblast Cell Migration through Rho A and Rho C Activation

This study investigated the roles of Rho protein in epidermal growth factor (EGF)-induced trophoblast cell migration and its mechanism. Using choriocarcinoma cell lines JEG-3 and JAR and first-trimester human chorionic villus explant cultures on matrigel, we examined EGF-mediated stimulation of trophoblast migration. EGF is shown to have a dose-dependent effect on trophoblast migration. A low concentration of EGF (1 ng/ml) has a stimulatory effect on cell migration, whereas high concentrations of EGF (100 ng/ml) shows an inhibitory effect. EGF (1 ng/ml) activates RhoA and RhoC, but not RhoB, through elevated protein levels and activity. EGF-induced migration was shown to be inhibited by either cell-permeable C3 exoenzyme transferase or selective RhoA or RhoC small interfering RNAs. The inhibition was not mitigated by the addition of EGF, suggesting that RhoA and RhoC play an important role in trophoblast migration and are obligatory for EGF action. Treatment of JEG-3 and JAR cells with RhoA small interfering RNA induced F-actin cytoskeleton disruption and cell shrinkage, which is consistent with the effect of C3 exoenzyme transferase, and this action was not mitigated by EGF treatment. RhoC small interfering RNA had no apparent effect on the F-actin arrangement, suggesting that RhoA but not RhoC takes part in the EGF-induced migration through F-actin rearrangement. These results indicate that RhoA and RhoC play more important roles than RhoB in EGF-mediated migration of trophoblast cells, and RhoA but not RhoC regulates this migration through F-actin cytoskeleton reorganization.

Strain‐Specific and Endocrine Control of Granular Convoluted Tubule Cells and Epidermal Growth Factor Expression in the Mouse Submandibular Gland

Submandibular glands (SMGs) of 11-week-old mice from four strains, ICR, C57BL/6J, BALB/c, and C3H/HeN were examined by immunohistochemistry for epidermal growth factor (EGF). In addition to sex-related differences in granular convoluted tubules (GCTs), the GCT cells were significantly larger in ICR mice than in other three strains. In males from each of the strains, almost all the GCT cells were strongly positive for EGF. The EGF-positive cells in the females, however, were markedly fewer in number, and were stained weaker in C57BL/6J, BALB/c, and C3H/HeN mice than in ICR mice. The GCT cells and their EGF expression in the F1 progeny from ICR and C3H/HeN strains were approximately intermediate between those of the parent strains of the same sex. T(3) and/or dihydrotestosterone (DHT) enhanced the GCT phenotype in the C3H/HeN mice, and remarkably increased the EGF-positive cells in females. Electron microscopy revealed that gold-labeling of EGF was confined to the secretory granules, and that the GCT cells in females, given T(3) + DHT, had a well-developed Golgi apparatus and net-like RER but few basal infoldings, whereas the equivalent cells in the untreated females had poor RER and prominent basal infoldings. These results suggest that the EGF concentration in SMGs is genetically high in ICR mice and low in other strain mice and that, considering the same response of GCT cells to T(3) and/or DHT between the high and low EGF strains, the low EGF concentrations might be partly caused by a lesser sensitivity of the GCT cells to thyroid hormones.

Protein kinase WNK2 inhibits cell proliferation by negatively modulating the activation of MEK1/ERK1/2

The recently identified subfamily of WNK protein kinases is characterized by a unique sequence variation in the catalytic domain and four related human WNK genes were identified. Here, we describe the cloning and functional analysis of the human family member WNK2. We show that the depletion of endogenous WNK2 expression by RNA interference in human cervical HeLa cancer cells led to the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases but, in contrast to the depletion of WNK1, had no effect on ERK5. Furthermore, expression of a kinase-dead WNK2-K207M mutant also activated ERK1/2 suggesting that WNK2 catalytic activity is required. Depletion of WNK2 expression increased G1/S progression and potentiated the cellular response to low epidermal growth factor concentrations. The molecular mechanism of ERK1/2 activation in WNK2-depleted cells lies downstream of the Raf kinases and involves MEK1 phosphorylation at serine 298 in both HeLa and HT29 colon cancer cells. This modification is linked to the upregulation of MEK1 activity toward ERK1/2. Together, these results provide evidence that WNK2 is involved in the modulation of growth factor-induced cancer cell proliferation through the MEK1/ERK1/2 pathway. The data identify WNK2 as a candidate tumor suppressor gene and suggest a coordinated activity of WNK kinases in the regulation of cell proliferation.

The effect of epidermal growth factor on matrix metalloproteinases and tissue inhibitors of metalloproteinase gene expression in cultured human gingival fibroblasts

Objective Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a role in the breakdown of the extracellular matrix during normal physiological processes, and in pathological processes, including periodontitis. The aim of this study was to evaluate the effect of epidermal growth factor (EGF) on the expression of MMPs and TIMPs in cultured human gingival fibroblasts. Methods Fibroblasts were stimulated with 10 −3, 10 −6 or 10 −12 M EGF for 24 h; untreated fibroblasts served as controls. Alterations in the expression of MMP-1, 2, 3, 7, 11, TIMP-1 and 2 were evaluated using real-time PCR and Western blotting. β-Actin expression was used as a reference to normalize gene expression. Results Increased MMP-1, 3, 7 and 11 expressions were observed at all EGF concentrations ( p < 0.05). At the lowest EGF concentration, MMP-1, 3 and 7 presented the lowest expression and MMP-11 presented the greatest expression; at higher EGF concentrations, MMP-1, 3 and 7 presented greater up-regulation, and MMP-11 lower up-regulation ( p < 0.05). Protein expression was similarly regulated by EGF: increased up-regulation of MMP-1, 3 and 7 was observed with increasing EGF concentrations, except for MMP-11 that exhibited greater up-regulation at the lower EGF concentration. The gene expression of MMP-2, TIMP-1 and 2 was not affected by EGF ( p < 0.05). Conclusions We conclude that EGF regulates expression for MMP-1, 3, 7 and 11 in a dose-dependent manner, suggesting that EGF may play a role in periodontal destruction and wound repair.

Epidermal growth factor (EGF) induces apoptosis in a transfected cell line expressing EGF receptor on its membrane

Interaction between epidermal growth factor (EGF) and EGF receptor (EGFR) promotes cell growth in most cell lines, but in a number of cell lines, EGF paradoxically inhibits proliferation. In the present study, we established a cell line expressing full-length human EGFR on membrane with a GFP fluorescence reporter at the C-terminal and studied the effects of EGF on cell proliferation in the transfected cell line. Our results suggested that low concentrations of EGF promoted proliferation, while high concentrations of EGF induced loss of adhesion, cell cycle arrest, apoptosis, and inhibition of proliferation. The effects of EGF on cell proliferation correlated well with the expression levels of EGFR. High concentrations of EGF induced both EGFR expression and apoptosis in a dose-dependent manner. Our study reported, for the first time, a relationship between the effects of EGF on cell proliferation and levels of EGFR expression in one cell line expressing different levels of EGFR caused by different concentrations of EGF treatment. The study should provide considerable insight into the effects of EGF on cell proliferation and tumor cell metastasis.

Synergistic activation of signalling to extracellular signal-regulated kinases 1 and 2 by epidermal growth factor and 4 beta-phorbol 12-myristate 13-acetate.

Signal transduction pathways are often embedded in complex networks, which result from interactions between pathways and feedback circuitry. In order to understand such networks, qualitative information on which interactions take place and quantitative data on their strength become essential. Here, we have investigated how the multiple interactions between the mitogen-activated protein kinase cascade and protein kinase C (PKC) affect the time profile of extracellular signal-regulated kinase (ERK) phosphorylation upon epidermal growth factor (EGF) stimulation in normal rat kidney fibroblasts. This profile is a major determinant for the cellular response that is evoked. We found that EGF stimulation leads to a biphasic ERK-PP pattern, consisting of an initial peak and a relaxation to a low quasi-steady state-phase. Costimulation with the EGF and PKC activator, 4 beta-phorbol 12-myristate 13-acetate (PMA) resulted in a similar pattern, but the ERK-PP concentration in the quasi-steady state-phase was synergistically higher than after stimulation with either EGF or PMA only. This resulted in prolonged signalling to ERK. PMA increased the EGF concentration sufficient to obtain half-maximum ERK phosphorylation. These data suggest that PKC amplifies EGF-induced signalling to ERK, without increasing its sensitivity to low EGF concentrations. Furthermore, PKC inhibition did not affect the ERK-PP time profile upon EGF stimulation and a cellular phospholipase A2 (cPLA(2)) inhibitor did not decrease the synergistic effect of EGF and PMA. This indicates that the positive feedback loop from ERK to Raf via cPLA(2) and PKC does not contribute significantly to signalling from EGF to ERK in normal rat kidney cells. Taken together, we provide a quantitative description of which reported interactions in this network affect the time profile of ERK phosphorylation.

The tethered configuration of the EGF receptor extracellular domain exerts only a limited control of receptor function.

Quantitative epidermal growth factor (EGF)-binding experiments have shown that the EGF-receptor (EGFR) is displayed on the surface of intact cells in two forms, a minority of high-affinity and a majority of low-affinity EGFRs. On the basis of the three-dimensional structure of the extracellular ligand binding domain of the EGFR, it was proposed that the intramolecularly tethered and autoinhibited configuration corresponds to the low-affinity receptor, whereas the extended configuration accounts for the high-affinity EGFRs on intact cells. Here we test this model by analyzing the properties of EGFRs mutated in the specific regions responsible for receptor autoinhibition and dimerization, respectively. Our results show that mutagenic disruption of the autoinhibitory tether in EGFR results in a decrease in the dissociation rate of EGF without a detectable change in EGFR activation and signaling through EGFR even in response to stimulation with low concentrations of EGF. Mutagenic disruption of the dimerization arm, on the other hand, increased the rate of EGF dissociation and impaired EGFR activation and signaling via the EGFR. This study demonstrates that the extended configuration of EGFR does not account for the apparent high-affinity EGF-binding to EGFR on intact cells. Furthermore, the autoinhibition conferred by the tethered configuration of the extracellular ligand-binding domain provides only a limited control of EGFR function.

EGF mediates multiple signals: dependence on the conditions

Epidermal growth factor (EGF) has been reported to both increase and decrease proliferation and apoptosis. In KB cells, EGF-induced alteration in proliferation and apoptosis are concentration and time dependent. High concentrations of EGF (10(-7) and 10(-8) M) stimulated proliferation and inhibited apoptosis at 24 h, with apoptosis increasing with prolonged exposure. Low concentrations of EGF (10(-10)-10(-11) M) inhibited apoptosis without affecting proliferation. EGFR protein expression was downregulated at high and upregulated at low EGF concentrations. Significant inverse correlation was shown between EGFR expression level and apoptosis. These results suggest the EGF receptor involvement in the modulation of apoptosis and the role of EGF anti-apoptotic effects in EGF-induced apoptosis.

Epidermal Growth Factor-induced Depletion of the Intracellular Ca2+ Store Fails to Activate Capacitative Ca2+Entry in a Human Salivary Cell Line

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca(2+) ([Ca(2+)](i)). Generally, agonist-mediated Ca(2+) mobilization involves both Ca(2+) release from internal stores and Ca(2+) influx activated by store depletion (i.e. capacitative or store-operated Ca(2+) influx). However, the role of capacitative Ca(2+) entry in EGF-mediated Ca(2+) mobilization is still largely unknown. In this study, we compared [Ca(2+)](i) signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca(2+) entry. Unlike carbachol and Tg, EGF (5 nm) elicited a transient [Ca(2+)](i) signal without a plateau phase in the presence of extracellular Ca(2+) and also failed to accelerate Mn(2+) entry. Repletion of extracellular Ca(2+) to cells stimulated with EGF in the absence of Ca(2+) elicited an increase in [Ca(2+)](i), indicating that EGF indeed stimulates Ca(2+) influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca(2+) release. Interestingly, the phospholipase C inhibitor completely inhibited Ca(2+) release induced by both EGF and carbachol and also reduced Ca(2+) influx responsive to carbachol but had no effect on Ca(2+) influx induced by EGF. EGF-induced Ca(2+) influx was potentiated by low concentrations (<5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca(2+) influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca(2+) influx responsive to carbachol but did not increase EGF-activated Ca(2+) influx. Both EGF and carbachol depleted internal Ca(2+) stores. Our results demonstrate that EGF-induced Ca(2+) release from internal stores does not activate capacitative Ca(2+) influx. Rather, EGF stimulates Ca(2+) influx via a mechanism distinct from capacitative Ca(2+) influx induced by carbachol and Tg.

Growth factor expression in cold and hot thyroid nodules.

Hot thyroid nodules (HTNs) are predominantly caused by constitutively activating thyrotropin receptor (TSHR) mutations leading to an activation of the cyclic adenosine monophosphate (cAMP)-cascade that stimulates growth and function of thyroid epithelial cells and confers growth advantage. In contrast to HTNs, the molecular etiology of szintigraphically cold thyroid nodules (CTNs) is largely unknown. An increased prevalence of toxic multinodular goiters in iodine-deficient regions has been reported. Growth factors increase during early stages of iodine deficiency in rats. These growth factors could modulate the proliferation of thyrocytes. In order to determine if and which growth factors could modulate the increase in thyroid epithelial cell proliferation in late stages of CTNs and HTNs we investigated epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and TGF-beta1 concentrations by enzyme-linked immunosorbant assay (ELISA) in CTNs (n = 7), HTNs (n = 9), and their normal surrounding tissue (ST). Insulin-like growth factor-1 (IGF-1) was determined in CTNs (n = 5) and HTNs (n = 10) and their surrounding tissues by radioimmunoassay (RIA). We found lower concentrations of all investigated growth factors and iodine in CTNs compared to surrounding normal tissues (ST). Only iodine showed a significant difference. Furthermore, we found significantly lower concentrations of EGF and TGF-beta1 concentration in HTNs compared to their STs. Differences of TGF-alpha and IGF-1 were not significant. In conclusion, low EGF, TGF-alpha, and IGF-1 concentrations in most CTNs in spite of low iodine concentrations argue against a pathophysiologic role of EGF, TGF-alpha, or IGF-1 in late stages of CTNs. The low EGF, TGF-alpha, and IGF-1 concentrations in HTNs irrespective of their clonal origin or the presence or absence of activating mutations argue for increased cAMP as the primary cause for thyroid epithelial cell proliferation in established HTNs. However, the pathophysiologic significance of low TGF-beta1 concentrations in CTNs and HTNs remains to be elucidated. It might be possible that growth factors like EGF, TGF-alpha, TGF-beta1, and IGF-1 play a more prominent role during early clonal expansion and that aberrant intrinsic signaling through a somatic mutation (e.g., TSHR for HTNs) confers the predominant selective growth advantage in later stages of HTNs or CTNs.

Angiogenic growth factors in human dentine matrix

The importance of growth factors in mediating the cellular responses to injury in the dentine–pulp complex is well recognized and several growth factors are reportedly sequestered in dentine matrix from where they may be released during repair processes. Local angiogenesis at the injury site appears to be critical for successful pulpal repair. Here, soluble and insoluble matrix fractions were isolated from human dentine and the amounts of several important angiogenic growth factors in these fractions measured by enzyme-linked immunosorbent assay (ELISA). The EDTA-soluble matrix fraction contained high concentrations of platelet-derived growth factor (PDGF-AB), lower concentrations of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and fibroblast growth factor (FGF2), and very low concentrations of epidermal growth factor (EGF). No FGF2 or PlGF could be detected in the insoluble matrix fractions, but these fractions contained some VEGF, lower concentrations of PDGF-AB and very low concentrations of EGF. It was concluded that dentine matrix contains angiogenic growth factors and that their release from the matrix after injury could make an important contribution to the overall reparative response of the dentine–pulp complex.