Cell-specific and developmental expression of phospholipase C-gamma and diacylglycerol in fetal lung

Epidermal growth factor (EGF) receptor (EGFR) regulates development of cell-cell communication in fetal lung, but the signal transduction mechanisms involved are unknown. We hypothesized that, in late-gestation fetal rat lung, phospholipase C-gamma (PLC-gamma) expression and activation by EGF is cell specific and developmentally regulated. PLC-gamma immunolocalized to cuboidal epithelium and mesenchymal clusters underlying developing saccules. PLC-gamma protein increased from day 17 to day 19 and then decreased. In cultured fetal lung fibroblasts, EGF stimulated PLC-gamma phosphorylation 2.6-fold (day 17), 10.8-fold (day 19), and 4.2-fold (day 21). EGF stimulated (3)H-labeled diacylglycerol production in fibroblasts (beginning on day 18 in female and on day 19 in male rats), but not in type II cells at any time during gestation. EGFR blockade abrogated the observed stimulation of PLC-gamma phosphorylation by EGF. In conclusion, PLC-gamma expression and activation by EGF in fetal lung are cell specific, corresponding to the development of EGFR expression. EGF induces diacylglycerol production in a cell- and gestation-specific manner. PLC-gamma activation by EGFR in fetal lung fibroblasts may be involved in EGF control of lung development.     

Dihydrotestosterone blocks fetal lung fibroblast-pneumonocyte factor at a pretranslational level

Fibroblast pneumonocyte factor (FPF) synthesis by fetal rat lung fibroblasts is augmented during gestation in the presence of cortisol. The control and cortisol-augmented levels of FPF production, as determined by FPF ability to stimulate saturated phosphotidylcholine synthesis by lung epithelial Type II cells, is delayed during development in fibroblasts derived from male fetuses as compared to those derived from female fetuses. The mechanism by which this delay occurs has been addressed. Pregnant rats treated in vivo with dihydrotestosterone (DHT) showed decreased FPF activity from control or cortisol-treated fibroblasts derived from 20-day-old male or female fetuses. In vitro translated proteins of size-fractionated lung RNA from 19-day-old fibroblasts that were pretreated with DHT in vitro showed decreased FPF activity compared to nontreated samples. This decreased FPF activity was present even if the DHT-pretreated cells were stimulated with cortisol prior to RNA preparation. Using a mouse model of testicular feminization that contains no receptors for androgens showed no change in the cortisol augmented FPF activity when the fibroblasts were pretreated with DHT. These data taken together suggest that the delayed FPF production of male-derived lung fibroblasts is a physiologic process which requires androgen receptors, and the mechanism by which androgens inhibit FPF production appears to affect events occurring mainly at a pretranslational level.     

Testosterone regulation of sex differences in fetal lung development.

Fetal lung development, in particular surfactant synthesis, exhibits a sexual dimorphism. Dihydrotestosterone (DHT) has been shown to delay fetal pulmonary surfactant production, but the potential role for testosterone is unknown. Both testosterone and DHT are potent masculinizing hormones, yet in some instances, an end organ specificity for DHT is present. We hypothesized that the delay in fetal lung surfactant production is dependent upon DHT such that inhibition of the synthesis of DHT from the precursor hormone testosterone would eliminate the sex difference by allowing the male fetus to produce surfactant at the female level. We tested this hypothesis using 17 beta-N,N-diethylcarbamoyl-4-aza-4-methyl-5-alpha-androstane-3-one (4-MA), a potent inhibitor of the enzyme 5 alpha-reductase, which converts testosterone into DHT. First, studies were performed in vivo. 4-MA (20 mg/kg/day) or an equivalent volume of vehicle was injected into pregnant rabbits from Day 12 through Day 26 of gestation. On Day 26, the fetuses were delivered, the lungs were lavaged, and fetal sex was noted. Treatment with 4-MA resulted in a lack of any male-female difference in the anogenital distance and no DHT was detected in the serum of any treated fetus. Phosphatidylcholine (PC), saturated phosphatidylcholine (SPC), and sphingomyelin (S) were measured in the lung lavage, and were expressed as the ratios of PC to sphingomyelin (PC:S) and SPC to sphingomyelin (SPC:S). Sex differences in the PC to sphingomyelin ratio of 4-MA-treated fetuses (female PC:S ratio, 1.43 +/- 0.14; male PC:S ratio, 1.00 +/- 0.13 [mean +/- SE]; P = 0.04) and in the SPC:S ratio of the 4-MA-treated group (female SPC:S ratio, 0.68 +/- 0.10; male SPC:S ratio, 0.35 +/- 0.10; P = 0.03) were present after treatment with 4-MA. The effect of testosterone and of 4-MA on fibroblast pneumonocyte factor (FPF) production was studied in vitro. Fetal rat lung fibroblasts were cultured to confluence with either no added androgen, DHT, testosterone, or testosterone plus 4-MA, and conditioned media for FPF were prepared. Conditioned media were added to fetal Type II cell cultures and FPF activity was measured as the degree of stimulation of the incorporation of [3H] choline into SPC. The conversion of radiolabeled testosterone to DHT by the fibroblasts was inhibited by 4-MA (10(-5) M). Conditioned media from untreated female fibroblasts stimulated with cortisol exhibited significant FPF activity ([3H]choline incorporation into SPC, 140 +/- 17% of control).(ABSTRACT TRUNCATED AT 400 WORDS)     

Sex-specific differences in rabbit fetal lung maturation in response to epidermal growth factor.

Males and females exhibit different stages of lung development at the same gestation with males lagging behind. We hypothesized that one of the mechanisms responsible for the sex-specific difference in fetal lung maturation is a delay in the onset of epidermal growth factor (EGF) activity in the male fetal lung. EGF influences growth and differentiation during development. We studied the effects of EGF on the incorporation of glycerol into lamellar body disaturated phosphatidylcholine (DSPC) in sex-specific fetal rabbit lung explants prepared at 21 and 24 days gestation (term 31 days). The explants were maintained in Waymouth's media + 10% stripped fetal calf serum with or without EGF (10 ng/ml). The incorporation of [1,3-14C]glycerol into lamellar body DSPC was assessed after 3, 5, or 7 days of culture. Female lung explants prepared at 21 days of gestation had increased incorporation of glycerol into DSPC over time in response to EGF treatment. Male lung explants prepared at 21 days did not respond to EGF treatment. In explants prepared at 24 days gestation, baseline glycerol incorporation into DSPC was higher in female as compared to male fetal lung explants. EGF-responsiveness was also sex-specific in these more mature explants, with the male explants now responding to EGF with a consistent increase in the incorporation of glycerol into lamellar body DSPC. We conclude that one of the mechanisms responsible for the lag in male fetal lung development is a delay in the onset of EGF activity.     

Development of fibroblast-type-II cell communications in fetal rabbit lung organ culture.

The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.     

Accelerated fetal lung maturation by estrogen is associated with an epithelial-fibroblast interaction

Summary The role of epithelial-mesenchymal interactions in the stimulation of lung development by estrogen is now investigated using organ cultures of lung from male and female fetal rats taken from Days 17 to 21 of gestation. Estradiol at 1 μg/ml was found to reduce cell proliferation in explants taken during a rapid growth phase (Day 18) and to stimulate surfactant synthesis in both males and females only in Day 20 explants when cell division is much slower. At this time more epithelial cells from estrogen-treated explants contained lamellar bodies, which were also secreted to fill the air sacs. These cultures also showed a significant increase in the frequency of cell-to-cell contacts between epithelial cells and fibroblasts. Uptake of tritiated estradiol by explants increased from Day 18 onward, and by autoradiography, labeling was located predominantly over fibroblasts. Using pure cultures of fetal and adult cells, uptake of labeled estradiol was significantly higher in fibroblasts than in corresponding epithelial cells, and estradiol did not directly enhance palmitate incorporation into epithelial cells. The results suggest that the earlier maturation and increased surfactant synthesis in female fetal lung is related at least in part to enhanced binding of estrogen by the fibroblast with subsequent transfer of a maturation factor to the fetal epithelium. This research project was supported by grants from the Medical Research Council of Canada and the Council for Tobacco Research, U.S.A., Inc.     

The effect of human urogastrone on lung phospholipids in fetal rabbits

Previous in vivo studies have demonstrated that mouse epidermal growth factor (EGF) can enhance fetal lung maturation. We have examined the effect of urogastrone, the human equivalent of mouse EGF and a related growth factor, on the phospholipid profile of fetal rabbit lung lavage and its action on fetal rabbit Type II pneumocytes in culture. Urogastrone (1 or 8 micrograms) given i.p. to fetal rabbits on day 25 of gestation resulted in increased total phospholipid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine contents, increased phosphatidylinositol and phosphatidylethanolamine as a proportion of phospholipid and decreased sphingomyelin as a proportion of phospholipid in lung lavages on day 28. These changes were unaccompanied by alterations in body weight or lung weight, DNA or protein concentrations. Urogastrone (16 micrograms) resulted in increased fetal deaths. Phospholipid profiles on day 27 were unchanged after fetal administration of urogastrone (1 microgram) on day 25. Urogastrone (0.01 and 0.1 ng/ml) added to fetal rabbit Type II pneumocytes in culture for 24 h enhanced the incorporation of radiolabelled choline and thymidine into phosphatidylcholine and DNA respectively. These findings indicate that human urogastrone can alter the phospholipid composition of the rabbit lung in a similar manner to that which occurs during maturation of the lung surfactant system in late pregnancy. This effect can be achieved, at least in part, by a direct action on Type II pneumocytes.     

The Effect of Corticosterone on the Growth of Perinatal Rat Lung Fibroblasts in Culture: Modulation by the Extracellular Matrix

Third passage lung fibroblasts from rat pups between the ages of fetal day 17 and postnatal day 15 were allowed to attach onto either tissue culture plastic or an endothelial cell-derived matrix, and were then exposed to different concentrations of corticosterone in culture medium containing 1% charcoal-stripped serum. The effect of the hormone on growth of these cells was assessed after 48 hours of exposure by radiothymidine incorporation into DNA. Lung fibroblasts on plastic responded to the hormone in an age-dependent manner; thus, cells which were obtained during late gestation (days 19–21) and after the third day of extrauterine life were consistently growth-inhibited by corticosterone, whereas those which were obtained from fetal donors prior to day 19 and from neonatal donors (days 0–3 after birth) were stimulated to grow in response to the hormone. On the other hand, cells that were plated onto an endothelial cell-derived matrix showed a different age-related response to the hormone. Thus, lung fibroblasts from all fetal donors and from postnatal donors up until 4–5 days of age were stimulated to grow in response to corticosterone. This modulatory effect of the matrix on the fibroblast response to corticosterone was also seen in early passage fibroblasts. In an attempt to identify the modulatory agent(s) in the extracellular matrix, fibroblasts from day 19 fetal rat lung were challenged with corticosterone in the presence of laminin, fibronectin, type IV collagen, type I collagen, heparin, or chondroitin sulfate. None of these agents exerted a modulatory effect resembling that seen with the extracellular matrix. These results suggest the existence during lung development of a fibroblast-endothelial interaction, the nature of which remains to be elucidated, which may serve to modify the effects of circulating hormones.     

Abrogation of apoptosis through PDGF-BB-induced sulfated glycosaminoglycan synthesis and secretion

Platelet-derived growth factor (PDGF)-BB-stimulated glycosaminoglycan (GAG) synthesis/secretion in fetal lung fibroblasts is dependent on sequential activation of the PDGF beta-receptor, phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt-1,2, and the GTPase Rab3D. Because the Akt pathway has been implicated in cell survival mechanisms, we investigated whether the pathway regulating GAG synthesis/secretion was antiapoptotic. PDGF-BB treatment protected fetal lung fibroblasts against serum starvation-induced apoptosis, whereas wortmannin, an inhibitor of PI3K, abrogated this protective effect. Transfection of constitutively active Akt into fetal lung fibroblasts also safeguarded the cells from apoptosis induced by serum starvation. To determine whether the antiapoptotic response was due, at least in part, to GAGs, we treated lung fibroblasts with beta-D-xyloside as well as with topically applied GAGs, specifically those produced by fetal lung fibroblasts. beta-D-xyloside increased GAG synthesis/secretion and diminished apoptosis. Application of sulfated GAGs, chondroitin sulfate, and heparan sulfate, but not nonsulfated hyaluronan, also resulted in diminished apoptosis. Moreover, topically applied sulfated GAGs increased Bcl-associated death promoter phosphorylation and diminished caspase-3 and -7 cleavage, indicating an antiapototic response. These data are compatible with the PDGF-BB-GAG signaling pathway regulating programmed fibroblast death in the fetal lung.     

Failure of hydrocortisone or growth factors to influence the senescence of fibroblasts in a new culture system for assessing replicative lifespan

It has been reported that the replicative lifespan of human fibroblasts can be substantially extended by supplementing the growth medium with hydrocortisone or increased levels of serum proteins. These observations have been made only on cell populations transferred many times at high cell density, and cumulative population doublings have been recorded, rather than a more direct measure of cell division potential. We have measured the replicative potential of human fibroblasts cultured so as to avoid conditions of high cell density, medium depletion, and departure from exponential growth. Two fetal lung and two newborn foreskin fibroblast strains were serially passaged in the presence or absence of hydrocortisone (HC), epidermal growth factor (EGF), and fibroblast growth factor (FGF) until they senesced. At each passage cells were plated at densities sufficiently low that colony-forming efficiency could be calculated. We determined cumulative population doublings and also estimated the number of cell generations attained under each condition. FGF caused small but possibly significant changes, while HC and EGF failed to substantially alter replicative lifespan. The reported effect of HC on the doubling potential of fetal lung fibroblasts is therefore not an inevitable action of this hormone on the senescence mechanism, but may instead depend for its apparent activity on the passage regimen used. The fibroblast's insensitivity to EGF as a modulator of replicative potential, as compared with the keratinocyte, whose lifespan can be tripled by EGF, implies that the mechanisms limiting the replicative potential of these two cell types are not identical.     

Hoxa-5 in mouse developing lung: cell-specific expression and retinoic acid regulation

Hoxa-5 is a homeobox gene that is highly expressed in the developing mouse lung. However, little is known about the molecular mechanisms controlling expression. We characterized the ontogeny of Hoxa-5 gene and protein expressions during lung development and then studied the cell-specific effects of retinoic acid (RA) on Hoxa-5 mRNA in fetal lung fibroblasts and MLE-12 mouse lung epithelial cells. Strong but constant Hoxa-5 gene and protein expressions were detected from mouse lung on embryonic day 13.5 to postnatal day 2. At baseline, the gene was strongly expressed in the fibroblasts of day 17.5 fetal mouse lungs. A very weak but reproducible expression was present in the MLE-12 cells. RA stimulated gene expression in both cell types in a time- and dose-dependent manner. Peak expression occurred much later in the MLE-12 cells compared with that in fibroblasts. Cycloheximide and actinomycin D treatment studies suggested that the differences in RA effect on each cell type may involve the presence of a repressor that can be overcome by RA.     

Lung-Derived Growth Factors: Ontogenic Shift in Parahormone Secretion in the Perinatal Rat Lung

Primary cultures of perinatal rat fibroblasts were found to produce at least three mitogenic activities which exhibited specificity for distinct cell types. One activity, lung growth factor(LGF), was a potent mitogen for chick embryonal fibroblasts, which also stimulated fetal rat lung fibroblasts to undergo DNA synthesis, provided that these cells were first exposed to a "competence" factor such as fibroblast growth factor or platelet-derived growth factor. Although LGF was active in the somatomedin-C (SmC) radioimmunoassay and resembled buffalo rat liver multiplication-stimulating activity (brlMSA) in molecular size, it appears to consist of a component that is neither SmC nor brlMSA. The second activity produced by perinatal rat lung cultures, pneumocyte-stimulating activity (PSA), stimulated mitosis in type II pneumocytes of postnatal rats, and was found to have physical attributes that are distinct from those of the other known pneumocyte-influencing factors. The third activity is a non-dialyzable substance which complements the mitogenic action of LGF on fetal lung fibroblasts, and appears to be a "competence" activity. An examination of the production of LGF and PSA by rat lung fibroblasts taken at various intervals of development revealed that fetal lung fibroblasts produce maximal levels of LGF but low levels of PSA, whereas, in neonatal lung fibroblasts, the situation is reversed. This ontogenic shift in the type of parahormone produced by the developing perinatal rat lung may be an important regulatory event in postnatal lung morphogenesis in this species.     

Effects of static magnetic fields on the growth of various types of human cells

The effects of a static magnetic field (SMF) on the proliferation of various types of human cells were determined. All cultures were maintained at 37 °C throughout the experiment. SMF was generated by placing two magnets oppositely oriented on either side of a T25 flask. The flux density in the flask ranged from 35 to 120 mT. Growth curves were constructed by plotting cell number at 18 h and 4, 7, 11, and 14 days after seeding, with the 18‐h point being a measure of attachment efficiency. Exposure to SMF significantly decreased initial attachment of fibroblasts and decreased subsequent growth compared to sham‐exposed control. Significant effects were observed in both fetal lung (WI‐38) and adult skin fibroblasts, but they were generally larger in the fetal lung fibroblast line. SMF did not affect attachment of human melanoma cells, but inhibited their growth by 20% on day 7. SMF produced no effects in a human adult stem cell line. Oxidant production increased 37% in WI‐38 cells exposed to SMF (230–250 mT) during the first 18 h after seeding, when cell attachment occurs. Conversely, no elevation in oxidant levels was observed after a prolonged 5‐day exposure. These results indicate that exposure to SMF has significant biological effects in some, but not all types of human cells. Bioelectromagnetics 32:140–147, 2011. © 2010 Wiley‐Liss, Inc.     

Appearance of functional EGF receptor kinase during rodent embryogenesis.

Mouse and rat embryonic tissues at various stages of development were examined for epidermal growth factor (EGF) receptor kinase activity. The phosphorylated EGF receptor from embryonic tissues appeared as a band of mol. wt. 170 000 daltons on SDS gels. It was clearly demonstrable in the developing mouse fetus from 10 days of gestation onwards. The distribution of the EGF receptor kinase was studied in various tissues of 13 day mouse fetuses. The activity was apparent in the skin, developing skeletal muscles and various internal organs but was notably absent in the liver and brain. The amnion was found to be one of the richest sources of activity while the yolk sac was negative, and the placenta was weakly positive. In 16 day rat fetuses the distribution was quite similar to that of the 13 day mouse fetus. The liver acquired EGF receptor kinase activity by 18 days of gestation and had high activity in neonates. Phosphoamino acid analysis revealed that phosphotyrosine was the major labelled amino acid residue in the embryonic tissues. Thus, the EGF receptor of fetal tissues as studied by immune precipitation and phosphorylation appears to be a similar entity to that found in adult mammalian tissues. This functional EGF receptor kinase activity could first be detected at the time of onset of organogenesis.     

Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice

We examined whether fibroblasts from subcutaneous, colon or lung tissues of nude mice influence the invasive potential of highly metastatic human colon carcinoma KM12SM cells. Primary cultures of nude mouse fibroblasts from skin, lung and colon were established. Invasive and metastatic KM12SM cells were cultured alone or with fibroblasts. Growth and invasive properties of the KM12SM cells were evaluated as well as their production of gelatinase activity. KM12SM cells were able to grow on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts. The conditioned media of KM12SM cells cocultured with skin, colon or lung fibroblasts were examined for the presence of type IV collagenase (gelatinase). KM12SM growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of 64 kDa type IV collagenase, whereas KM12SM cells cocultivated with nude mouse skin fibroblasts did not. In contrast, human squamous cell carcinoma A431 cells produced significant levels of collagenase type IV when cocultured with nude mouse skin fibroblasts, a tissue they invaded and completely penetrated. Incubation of KM12SM cells in serum-free medium containing recombinant human interferon-beta (fibroblast interferon) was associated with significant reduction in gelatinase activity. Since the production of type IV collagenase by human colon cancer cells is specifically inhibited by mouse skin fibroblasts but not by colon or lung fibroblasts the data suggest that organ-specific fibroblasts can influence the invasive and metastatic properties of KM12SM cells.     

表皮生长因子对鼠胚成纤维细胞细胞周期的调节作用

本文观察了表皮生长因子（ＥＧＦ）对小鼠胚胎成纤维细胞Ｃ_３Ｈ／１０Ｔ_１／２ＣＬ_８（简称Ｃ_３Ｈ／１０）细胞周期的影响。结果表明：ＥＧＦ使Ｓ期提前,细胞周期缩短。进一步探讨了ＥＧＦ对细胞周期影响的机制,发现ＥＧＦ可活化在细胞周期调节中起重要作用的Ｐ３４~（ｃｄｃ２）激酶（简称ＣＤ２Ｋ）,使ＣＤ２Ｋ在周期中活性高峰出现的时间提前,提示ＥＧＦ对细胞周期的影响可能通过作用于ＣＤ２Ｋ实现的。     

Hormonal control of the replication of human fetal fibroblasts: role of somatomedin C/insulin-like growth factor I

Sparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin-like growth factor I (SM-C/IGF-I). At both developmental stages, the addition of SM-C/IGF-I (100 ng/ml) increased cell number at day 3 1.4-fold in serum-free medium and 2-fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose-response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth-promoting effects of SM-C/IGF-I, with a half-maximal response occurring at 6 ng/ml SM-C/IGF-I. This biological action of SM-C/IGF-I correlated with SM-C/IGF-I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM-C/IGF-I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM-C/IGF-I in stimulating replication of postnatal fibroblasts. The combination of SM-C/IGF-I (100 ng/ml), dexamethasone (10(-7) M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM-C/IGF-I and dexamethasone in fetal fibroblasts. In fetal cells, SM-C/IGF-I + EGF + PDGF +/- dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50-100% more responsive than postnatal cells to the proliferative effect of serum.     

Extracellular regulated kinase5 is expressed in fetal mouse submandibular glands and is phosphorylated in response to epidermal growth factor and other ligands of the ErbB family of receptors

Growth factors and their receptors regulate development of many organs through activation of multiple intracellular signaling cascades including a mitogen‐activated protein kinase (MAPK). Extracellular regulated kinases (ERK)1/2, classic MAPK family members, are expressed in fetal mouse submandibular glands (SMG), and stimulate branching morphogenesis. ERK5, also called big mitogen‐activated protein kinase 1, was recently found as a new member of MAPK super family, and its biological roles are still largely unknown. In this study, we investigated the expression and function of ERK5 in developing fetal mouse SMGs. Western blotting analysis showed that the expression pattern of ERK5 was different from the pattern of ERK1/2 in developing fetal SMGs. Both ERK1/2 and ERK5 were phosphorylated after exposure to ligands of the ErbB family of receptor tyrosine kinases (RTKs). Phosphorylation of ERK1/2 was strongly induced by epidermal growth factor (EGF) in SMG rudiments at embryonic day 14 (E14), E16 and E18. However, ERK5 phosphorylation induced by EGF was clearly observed at E14 and E16, but not at E18. Branching morphogenesis of cultured E13 SMG rudiments was strongly suppressed by administration of U0126, an inhibitor for ERK1/2 activation, whereas the phosphorylation of ERK5 was not inhibited by U0126. BIX02188, a specific inhibitor for ERK5 activation, also inhibited branching morphogenesis in cultured SMG rudiments. These results show that EGF‐responsive ERK5 is expressed in developing fetal mouse SMG, and suggest that both ERK1/2 and ERK5 signaling cascades might play an important role in the regulation of branching morphogenesis.