Serum-free Primary Culture Research Articles

Tonic ErbB signaling underlies TGFβ-induced activation of ERK and is required for lens cell epithelial to myofibroblast transition.

Fibrosis is a major, but incompletely understood, component of many diseases. The most common vision-disrupting complication of cataract surgery involves differentiation of residual lens cells into myofibroblasts. In serum-free primary cultures of lens epithelial cells (DCDMLs), inhibitors of either ERK or of ErbB signaling prevent TGFβ from upregulating both early (fibronectin) and late (αSMA) markers of myofibroblast differentiation. TGFβ stimulates ERK in DCDMLs within 1.5 h. Kinase inhibitors of ErbBs, but not of several other growth factor receptors in lens cells, reduce phospho ERK to below basal levels in the absence or presence of TGFβ. This effect is attributable to constitutive ErbB activity playing a major role in regulating the basal levels pERK. Additional studies support a model in which TGFβ-generated reactive oxygen species serve to indirectly amplify ERK signaling downstream of tonically active ErbBs to mediate myofibroblast differentiation. ERK activity is in turn essential for expression of ErbB1 and ErbB2, major inducers of ERK signaling. By mechanistically linking TGFβ, ErbB, and ERK signaling to myofibroblast differentiation, our data elucidate a new role for ErbBs in fibrosis and reveal a novel mode by which TGFβ directs lens cell fate.

Culture of Mouse Thymic Epithelial Cells in Serum-Free Medium.

Primary cell culture systems are widely used as a valuable method for analyzing the biological functions of specific cells in vitro. Recently, various serum-free primary cell culture methods have been developed that do not involve the use of animal serums. Since the thymus is comprised of many cell types, such as thymocytes, thymic epithelial cells, macrophages, and fibroblasts, thymic epithelial cells must be isolated for their functional analysis in vitro. This chapter describes the detailed protocol for the selective primary culture of thymic epithelial cells using defined serum-free medium.

Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass clearance

BackgroundMicroglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood.MethodsTo understand how microglia respond during demyelination, we fed mice cuprizone—a potent demyelinating agent—and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response.ResultsThe cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination.ConclusionsMicroglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state.

Effects of Fatty Acids on Proliferation of Cultured Wild-type and FABP5-KO Thymic Epithelial Cells.

Lipids including fatty acids (FAs), which are water-insoluble molecules, are not only a cellular energy source but also signaling molecules that induce and modulate the expression of various cellular functions. Fatty acid-binding proteins (FABP) bind FAs in the cytoplasm, and are thought to determine the cellular localization of FAs. In a previous observation, FABP5 was expressed in thymic epithelial cells (TEC) in the thymus and was influenced by FAs. Fatty acids have mostly inhibitory effects on various cell types, including cancer cells, but their effects on TEC have not been well investigated. In this study, we investigated the effects of long-chain FAs (LCFAs) and the involvement of FABP5 in cell proliferation using a serum-free primary culture system. The results showed that saturated fatty acids did not affect proliferation, but n-3 long-chain polyunsaturated FA (LCPUFA) reduced, n-6 LCPUFA increased, and retinoic acid strongly reduced the percentage of proliferating wild-type TEC. The proliferation of FABP5-KO TEC was more significantly affected by LCPUFA, suggesting that FABP5 is an important modulator of FA-mediated TEC proliferation. These observations may provide a basis for exploring the properties of TEC.

Mild Traumatic Brain Injury/Concussion Initiates an Atypical Astrocyte Response Caused by Blood-Brain Barrier Dysfunction.

Mild traumatic brain injury/concussion (mTBI) accounts for 70-90% of all reported TBI cases and causes long-lasting neurological consequences in 10-40% of patients. Recent clinical studies revealed increased blood-brain barrier (BBB) permeability in mTBI patients, which correlated with secondary damage after mTBI. However, the cascade of cellular events initiated by exposure to blood-borne factors resulting in sustained damage is not fully understood. We previously reported that astrocytes respond atypically to mTBI, rapidly losing many proteins essential to their homeostatic function, while classic scar formation does not occur. Here, we tested the hypothesis that mTBI-induced BBB damage causes atypical astrocytes through exposure to blood-borne factors. Using an mTBI mouse model, two-photon imaging, an endothelial cell-specific genetic ablation approach, and serum-free primary astrocyte cultures, we demonstrated that areas with atypical astrocytes coincide with BBB damage and that exposure of astrocytes to plasma proteins is sufficient to initiate loss of astrocyte homeostatic proteins. Although mTBI resulted in frequent impairment of both physical and metabolic BBB properties and leakage of small-sized blood-borne factors, deposition of the coagulation factor fibrinogen or vessel rupture were rare. Surprisingly, even months after mTBI, BBB repair did not occur in areas with atypical astrocytes. Together, these findings implicate that even relatively small BBB disturbances are sustained long term, and render nearby astrocytes dysfunctional, likely at the cost of neuronal health and function.

Fibronectin regulates growth factor signaling and cell differentiation in primary lens cells.

Lens epithelial cells are bound to the lens extracellular matrix capsule, of which laminin is a major component. After cataract surgery, surviving lens epithelial cells are exposed to increased levels of fibronectin, and so we addressed whether fibronectin influences lens cell fate, using DCDML cells as a serum-free primary lens epithelial cell culture system. We found that culturing DCDMLs with plasma-derived fibronectin upregulated canonical TGFβ signaling relative to cells plated on laminin. Fibronectin-exposed cultures also showed increased TGFβ signaling-dependent differentiation into the two cell types responsible for posterior capsule opacification after cataract surgery, namely myofibroblasts and lens fiber cells. Increased TGFβ activity could be identified in the conditioned medium recovered from cells grown on fibronectin. Other experiments showed that plating DCDMLs on fibronectin overcomes the need for BMP in fibroblast growth factor (FGF)-induced lens fiber cell differentiation, a requirement that is restored when endogenous TGFβ signaling is inhibited. These results demonstrate how the TGFβ-fibronectin axis can profoundly affect lens cell fate. This axis represents a novel target for prevention of late-onset posterior capsule opacification, a common but currently intractable complication of cataract surgery.

Rolipram potentiates bevacizumab-induced cell death in human glioblastoma stem-like cells

AimsGlioblastoma cancer stem-like cells (GCSCs) promote themselves proliferation by secreting the vascular endothelial growth factor A (VEGFA) in an autocrine manner, positively regulated by phosphodiesterase IV (PDE4). In the current study, we investigated the putative cytotoxic effect of bevacizumab, a VEGFA blocker, alone and in combination with a specific inhibitor of PDE4 called rolipram on GCSCs isolated from human surgical tumor specimen with a focus on PI3K/AKT pathway. Main methodsCD133+/CD15+ GCSCs were characterized by flow cytometry and expanded in a serum-free primary culture system. The cell survival, apoptosis, and protein expression values were measured using MTT assay, TUNEL staining and western blot, successively. Intracellular cAMP and free secreted VEGFA levels were assessed by cAMP enzyme immunoassay and ELISA, respectively. Key findingsBevacizumab suppressed GCSCs survival with IC50~6.5μg/ml and enhanced the levels of apoptosis, p53 and cleaved-caspase3 along with a decrease in free VEGFA levels and ERKs activation. However, there was no significant modulation of AKT phosphorylation on serine 473, the intracellular PDE4A, VEGFA and cAMP levels. More cytotoxicity in co-treated cells coupled with a more substantial decline in the free VEGFA levels and a greater increase in the quantities of p53 and cleaved-caspase3 compared to those treated with bevacizumab alone. Co-treatment reduced phospho-AKT, endogenous VEGFA and PDE4A values but elevated cAMP levels. SignificanceThis study highlighted a booster cytotoxic effect of combined rolipram and bevacizumab treatment on the GCSCs primary culture, suggesting that this approach is warranted in treatment of GBMs overexpressing VEGFA and PDE4A.

Acquisition of tumorigenic potential and enhancement of angiogenesis in pulmonary stem/progenitor cells through Oct-4 hyperexpression.

Cancer stem cells, also known as cancer initiating cells (CICs), are considered to be responsible for tumor growth and chemoresistance. Different hypotheses have been proposed to explain the origin of CICs, including mutations in adult stem/progenitor cells or the acquisition of stem-like characteristics in differentiated cells; however, studies have yielded conflicting identification for CICs and have little information for the origin to generate CICs. Part of the difficulty in identifying CICs may stem from the fact that the CICs studied have been largely derived from cancer cell lines or well-developed tumors. In previous studies, we have reported the enrichment of mouse pulmonary stem/progenitor cells (mPSCs) by using serum-free primary selection culture followed by FACS isolation using the coxsackievirus/adenovirus receptor (CAR) as the positive selection marker. Here, we demonstrated that overexpression of the pluripotent transcription factor Oct-4 is sufficient to induce CAR+/mPSCs transformation, which we name CAR+/mPSCsOct-4_hi. These transformed cells possess cancer initiating and chemoresistance potential, as well as exhibiting remarkable expression of certain proangiogenic factors, including angiopoietins (ANGs) and VEGF, and enhanced angiogenic potential. Moreover, CAR+/mPSCsOct-4_hi actively participated in tumor blood vessel formation and triggered a novel angiogenic mechanism, the angiopoietins/Tie2 signaling pathway. These study provide critical evidence supporting the possible origin to generate CICs, and help elucidate the pathways responsible for CICs-mediated blood vessel formation.

L-tyrosine induces melanocyte differentiation in novel pink-eyed dilution castaneus mouse mutant showing age-related pigmentation

BackgroundThe mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2p) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (pcas/Oca2p−cas) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2p−cas usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging. ObjectiveThe aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system. MethodsThe characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD). ResultsThe proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes. ConclusionThese results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the mechanisms of age-related pigmentation in human skin, such as melasma and solar lentigines.

Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells.

Fibroblast growth factors (FGFs) play a central role in two processes essential for lens transparency--fiber cell differentiation and gap junction-mediated intercellular communication (GJIC). Using serum-free primary cultures of chick lens epithelial cells (DCDMLs), we investigated how the FGF and bone morphogenetic protein (BMP) signaling pathways positively cooperate to regulate lens development and function. We found that culturing DCDMLs for 6 d with the BMP blocker noggin inhibits the canonical FGF-to-ERK pathway upstream of FRS2 activation and also prevents FGF from stimulating FRS2- and ERK-independent gene expression, indicating that BMP signaling is required at the level of FGF receptors. Other experiments revealed a second type of BMP/FGF interaction by which FGF promotes expression of BMP target genes as well as of BMP4. Together these studies reveal a novel mode of cooperation between the FGF and BMP pathways in which BMP keeps lens cells in an optimally FGF-responsive state and, reciprocally, FGF enhances BMP-mediated gene expression. This interaction provides a mechanistic explanation for why disruption of either FGF or BMP signaling in the lens leads to defects in lens development and function.

Astrocytes and microglia but not neurons preferentially generate N-terminally truncated Aβ peptides

The neuropathological hallmarks of Alzheimer's disease include extracellular neuritic plaques and neurofibrillary tangles. The neuritic plaques contain β-amyloid peptides (Aβ peptides) as the major proteinaceous constituent and are surrounded by activated microglia and astrocytes as well as dystrophic neurites. N-terminally truncated forms of Aβ peptides are highly prevalent in neuritic plaques, including Aβ 3-x beginning at Glu eventually modified to pyroglutamate (Aβ N3pE-x), Aβ 2-x, Aβ 4-x, and Aβ 5-x. The precise origin of the different N-terminally modified Aβ peptides currently remains unknown. To assess the contribution of specific cell types to the formation of different N-terminally truncated Aβ peptides, supernatants from serum-free primary cell cultures of chicken neurons, astrocytes, and microglia, as well as human astrocytes, were analyzed by Aβ-ELISA and one- and two-dimensional SDS-urea polyacrylamide gel electrophoresis followed by immunoblot analysis. To evaluate the contribution of β- and γ-secretase to the generation of N-terminally modified Aβ, cultured astrocytes were treated with membrane-anchored “tripartite β-secretase (BACE1) inhibitors” and the γ-secretase inhibitor DAPT. Neurons, astrocytes, and microglia each exhibited cell type-specific patterns of secreted Aβ peptides. Neurons predominantly secreted Aβ peptides that begin at Asp1, whereas those released from astrocytes and microglia included high proportions of N-terminally modified Aβ peptides, presumably including Aβ 2/3-x and 4/5-x. The inhibition of BACE1 reduced the amount of Aβ 1-x in cell culture supernatants but not the amount of Aβ 2-x.

Bat airway epithelial cells: a novel tool for the study of zoonotic viruses.

Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.

Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1–100 ng/ml) suppressed androstenedione production. Inhibin (1–100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: The influence of extracellular matrix on intramuscular and extramuscular adipogenesis1

The extracellular matrix (ECM) and specific ECM components can have a major influence on cell growth, development, and phenotype. The influence of the ECM and ECM components on adipogenesis in vivo and in vitro will be reviewed in this paper. Engelbreth-Holm-Swarm substratum and laminin per se markedly increased attachment, spreading, and hypertrophy of preadipocytes in serum-free primary cultures of porcine adipose tissue stromal-vascular cells. Furthermore, primary cultures of stromal-vascular cells showed that preadipocytes express ECM components after preadipocyte recruitment. Staining for plant lectins, type IV collagen, and laminin in fetal pig adipose tissue demonstrates that adipocyte reactivity for laminin was strong throughout fetal development and was similar for developing adipocytes and vasculature. However, lectin binding and type IV collagen reactivity of blood vessels preceded that for adipocytes. Therefore, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Specific inhibitors and modulators of collagen synthesis have been used to evaluate the role of collagens in the differentiation of bovine intramuscular preadipocytes (BIP) and other preadipocyte cell lines. Triglyceride accretion of BIP cells was inhibited by a general inhibitor of collagen biosynthesis, whereas specific inhibitors or modulators of type IV collagen inhibited 3T3-L1 cell differentiation. Further study revealed that compared with collagens types I to IV, type V and VI collagens have an important and active role in BIP adipogenesis. The growth of intramuscular bovine adipose tissue may be dependent on collagen newly synthesized and organized by the adipocytes per se. The role of extracellular or ECM proteolysis in regulating adipogenesis also will be reviewed in this paper. Many members of the matrix metalloproteinase (MMP) family are expressed by adipocytes, and specific inhibition of MMP-9 greatly reduces adipogenesis in vitro. Possibly, MMP and other proteases regulate turnover of key adipocyte ECM proteins that are involved in the regulation of preadipocyte proliferation and differentiation.

In-vitro effects of the antimicrobial peptide Ala8,13,18-magainin II amide on isolated human first trimester villous trophoblast cells

BackgroundResearch on antimicrobial cationic peptides (AMPs) has gained pace toward using their potential to replace conventional antibiotics. These peptides preferentially interact with negatively charged membrane lipids typically seen in bacteria and thereby lead to membrane perturbations and membrane dysfunction. However, one possible disadvantage of AMP drugs is their potential for toxicity, especially to those cells which display externalization of negatively charged moieties to the outer leaflet of the plasma membrane during the process of syncytialization. Human placental villous trophoblast is one such cell type. Indeed, intra-vaginal administration of an antimicrobial cationic peptide Ala8,13,18-magainin II amide (AMA) which is a synthetic analogue of magainin 2 derived from Xenopus frog has been observed to result in inhibition of pregnancy establishment in monkeys. However, only little is known about the cellular behavior of early placental cytotrophoblasts (CTB) in the presence of cationic antimicrobial peptides. It is believed that suitable cell culture approaches using AMA as a representative alpha-helical AMP may yield tangible knowledge in this regard.MethodsImmunocytochemical (ICC) analyses using confocal microscopy (n = 6 for each treatment sub-group) and Western blot (WB) method (n = 5 for each treatment sub-group) of CTB differentiation based on synthesis of beta-hCG and hPL, and apoptosis based on apoptosis-associated cytokeratin 18 neo-epitope (CK18f) were performed for CTB isolated from human first trimester placental villi and grown in serum-free primary culture for 24 h, 48 h and 96 h on rat-tail collagen with and without AMA (1000 ng/ml). Moreover, secretion of beta-hCG and hPL into conditioned media from isolated CTB grown in vitro for 24 h, 48 h and 96 h (n = 6/each sub-group) with and without AMA was examined using enzyme immunoassays. Furthermore, TUNEL assay, and cell viability based on LDH leakage into medium (n = 6/each sub-group) were assessed to examine the phenomenon of cell death with time and administration of AMA.ResultsCTB in serum-free primary culture showed increased (P < 0.05) level of synthesis and secretion of beta-hCG and hPL with time, and higher (P < 0.05) level of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h of culture. Exposure of CTB to AMA resulted in lower (P < 0.05) level of synthesis and secretion of beta-hCG and hPL, as well as, an increase (P < 0.05) of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h as compared to the control treatment.ConclusionsAdministration of AMA resulted in attenuation of differentiation, enhancement in apoptosis and loss of viability in early placental villi trophoblast cells in primary culture. Thus, it appears that administration of alpha-helical AMP may adversely affect the process of placentation and pregnancy outcome.

Inhibitory Effects of Dexamethasone on Epidermal Growth Factor-Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes

We investigated the effects of dexamethasone on epidermal growth factor (EGF)-induced DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. Isolated hepatocytes were cultured at a density of 3.3 × 10⁴ cells/cm² in Williams' medium E containing 5% bovine calf serum and various concentrations of dexamethasone for 1, 2 and 3 h. After the 3-h attachment period, the medium was changed, and cells were cultured in serum-free and dexamethasone-free Williams' medium E with or without glucocorticoid receptor antagonists. The growth-stimulating effects of EGF (20 ng/ml) on the primary cultured hepatocytes were time- and concentration-dependently inhibited by dexamethasone added to the culture medium. The mineral corticoid aldosterone (10⁻⁷ M) did not produce the same growth-inhibitory effects as dexamethasone (10⁻⁸ M). The inhibitory effects of dexamethasone were reversed by treatment with the glucocorticoid receptor antagonist mifepristone (RU486, 10⁻⁶ M) or a monoclonal antibody against glucocorticoid receptor (100 ng/ml). In addition, the growth-inhibitory effects of dexamethasone did not affect EGF-induced p42 mitogen-activated protein (MAP) kinase phosphorylation. These results indicate that dexamethasone concentration-dependently delays and inhibits the EGF-induced DNA synthesis and proliferation through its own intracellular receptor in primary cultures of adult rat hepatocytes.

Inhibitory Effects of Dexamethasone on Hepatocyte Growth Factor–Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes

We investigated the effects of dexamethasone on hepatocyte growth factor (HGF)-induced DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. Isolated hepatocytes were cultured at a density of 3.3 × 104 cells/cm2 in Williams’ medium E containing 5% newborn bovine serum and various concentrations of dexamethasone for 1, 2, and 3 h. After a 3-h attachment period, the medium was then changed, and cells were cultured in serumfree dexamethasone (10−10 M)-containing Williams’ medium E with or without glucocorticoid receptor antagonists. After addition of dexamethasone to the culture medium, the growth-stimulating effects of HGF (5 ng/mL) on the primary cultured hepatocytes were time- and dose-dependently inhibited. The mineralcorticoid aldosterone (10−7 M) did not produce the same growth-inhibitory effects as dexamethasone (10−8 M). The inhibitory effects of dexamethasone were reversed by treatment with the glucocorticoid-receptor antagonist mifepristone (RU486, 10−6 M) or a monoclonal antibody against glucocorticoid receptor (100 ng/mL). In addition, the growth-inhibitory dose of dexamethasone did not affect HGF-induced receptor tyrosine kinase and extracellular signal-regulated kinase 2 phosphorylation. These results indicate that dexamethasone dose-dependently delays and inhibits HGF-induced DNA synthesis and proliferation through its own intracellular receptor in primary cultures of adult rat hepatocytes.

Regulation of Lens Gap Junctions by Transforming Growth Factor Beta

Gap junction-mediated intercellular communication (GJIC) is essential for the proper function of many organs, including the lens. GJIC in lens epithelial cells is increased by FGF in a concentration-dependent process that has been linked to the intralenticular gradient of GJIC required for lens transparency. Unlike FGF, elevated levels of TGF-beta are associated with lens dysfunction. We show that TGF-beta1 or -2 up-regulates dye coupling in serum-free primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) via a mechanism distinct from that utilized by other growth factors. Remarkably, the ability of TGF-beta and of FGF to up-regulate GJIC is abolished if DCDMLs are simultaneously exposed to both factors despite undiminished cell-cell contact. This reduction in dye coupling is attributable to an inhibition of gap junction assembly. Connexin 45.6, 43, and 56-containing gap junctions are restored, and intercellular dye coupling is increased, if the activity of p38 kinase is blocked. Our data reveal a new type of cross-talk between the FGF and TGF-beta pathways, as well as a novel role for TGF-beta and p38 kinase in the regulation of GJIC. They also provide an explanation for how pathologically increased TGF-beta signaling could contribute to cataract formation.

Interleukin-1α Stimulates the Differentiation of Melanocytes but Inhibits the Proliferation of Melanoblasts from Neonatal Mouse Epidermis

Interleukin (IL)-1alpha is one of the important cytokines involved in regulating immunological reactions in the mouse skin. However, it is not known whether IL-1alpha regulates the proliferation and differentiation of mouse epidermal melanocytes. In this study, to investigate the role of IL-1alpha in the regulation of the proliferation and differentiation of mouse epidermal melanocytes, IL-1alpha was supplemented to serum-free primary cultures of epidermal cell suspensions from the initiation of the primary culture (keratinocytes and melanoblasts-melanocytes) as well as to pure cultures of melanoblasts-melanocytes (keratinocyte-depleted cultures, after 14 days), and its effect was tested. IL-1alpha inhibited the proliferation of undifferentiated melanoblasts irrespective of the presence or absence of keratinocytes, whereas the cytokine inhibited the proliferation of differentiated melanocytes only in the presence of keratinocytes. Moreover, IL-1alpha induced the differentiation of melanocytes and, in addition, stimulated tyrosinase activity, melanin synthesis, and dendritogenesis of melanocytes irrespective of the presence or absence of keratinocytes. These results suggest that IL-1alpha is involved in inhibiting the proliferation of neonatal murine epidermal melanoblasts and in stimulating the differentiation, melanogenesis, and dendritogenesis of melanocytes. The results also suggest that IL-1alpha inhibits the proliferation of differentiated melanocytes in cooperation with keratinocyte-derived factors.

Insulin increases the expression of contractile phenotypic markers in airway smooth muscle

We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 microM) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70(S6K) (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 microM) or the PI 3-kinase inhibitor LY-294002 (10 microM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus.