Biomedical subjects
Publications and source records attributed to S L Katyal.
Clara cells are nonciliated, nonmucous, secretory cells of the pulmonary airways. These cells are known to secrete a variety of proteins, including Clara cell 10-kDa protein/uteroglobin. This protein consists of a homodimer of 70-77 amino acid polypeptides arranged in antiparallel fashion. In vitro testing suggests that the protein suppresses inflammation. The physiologic role of the protein remains to be determined.
In this study, we examined the effects of dexamethasone (DEX) on airway branching and subsequent lung maturation. DEX treatment of fetal rat lung explants was initiated during the early pseudoglandular stage of development. Day 14 fetal lung explants were cultured with and without DEX for 4 d. Explants treated with 10 nM or higher concentrations of DEX showed features of both distorted and accelerated maturation. DEX-treated lungs had growth retardation, distorted branching, dilated proximal tubules, and suppressed proliferation of epithelial cells of the distal tubules. Several biochemical and morphologic features of accelerated maturation were also observed: 1) the epithelial cells lining the distal tubules (prospective respiratory airways) were generally cuboidal or flattened; 2) the cuboidal cells often contained lamellar bodies and abundant glycogen; 3) rudimentary septa and large airspace were present; 4) mesenchymal tissue was attenuated and compressed between adjacent epithelial tubules; 5) the distribution of SP-C mRNA in distal tubules was more mature, with individual and clusters of cells expressing SP-C transcripts; and 6) the transcript levels of several genes related to epithelial growth [keratinocyte growth factor (KGF), KGF receptor, and hepatocyte growth factor receptor] and differentiation [surfactant proteins, SP-A, SP-B and SP-C and the Clara cell secretory protein, CC10] were precociously increased. These results show that DEX treatment of the lung during the early pseudoglandular stage accelerates the acquisition of several features of advanced maturation that normally accompany late stages of fetal development. We postulate that KGF mediates at least some effects of DEX on lung maturation and gene expression.
We have reported that dexamethasone (DEX) treatment of early embryonic rat lungs in culture induced features of both distorted and accelerated maturation. In this report, we investigated the effects of retinoids on normal and DEX-induced lung development in vitro. Lung maturation was assessed by examining the morphology and the expression of genes related to epithelial differentiation (surfactant proteins, SP-A, SP-B and SP-C and Clara cell protein, CC10) and growth [keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF)]. We cultured d 14 and 15 fetal rat lungs in the presence of DEX (1-1000 nM) and/or all-trans-retinoic acid (RA) (10(-7)-10(-5) M) for 4 d. RA at 10(-6) and 10(-5) M inhibited branching and dilated the distal tubules, and at 10(-5) M caused dilatation of the proximal tubules destined to form the trachea and the main bronchi. The adverse effects of DEX, such as distorted branching, tubular dilatation, and suppression of both lung growth and epithelial cell proliferation, were all prevented by RA. In addition, RA inhibited several features of DEX-induced accelerated maturation, such as: 1) the increased levels of SP-A, SP-B, and CC10 mRNAs; 2) the attenuation of mesenchymal tissue; and 3) the mature distribution of cells expressing SP-C mRNA. In contrast, RA potentiated the increase of KGF and decrease of HGF transcripts induced by DEX. In conclusion, the study shows antagonism by RA of DEX-induced effects on lung morphology and gene expression. We postulate that normal lung development requires a balanced action of endogenous retinoids and glucocorticoids.
We recently suggested that peroxisome proliferators (PPs), 3,3',5-triiodo-L-thyronine (T3), and 9-cis retinoic acid (9-cis RA) induce hepatocyte proliferation in rats through the activation of their nuclear receptors, PP-activated receptors, T3 receptors, and retinoid X receptors. To test whether nuclear hormone receptor-mediated cell proliferation can be observed in organs other than liver, we examined the effects of these agents on the pancreas and kidneys of male Wistar rats using BrdUrd immunohistochemistry. A single s.c. injection of T3 (2 mg/kg) and single intragastric administration of 9-cis RA (40 mg/kg) or 4-chloro-6-(2, 3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide (200 mg/kg) induced a wave of DNA synthesis in the pancreatic acinar cells and in the proximal tubular epithelial cells of the kidneys, peaking after 24 h. No stimulation of DNA synthesis was observed in ductal or islet cells of the pancreas and in glomeruli of the kidneys. All-trans-retinoic acid, a ligand for retinoic acid receptor, at a dose (200 mg/kg) that induced hepatocyte proliferation, had no effects on cell proliferation of the pancreas and the kidneys. The results suggest that T3, 9-cis RA, and PP activate genes that regulate cell proliferation in target cells through receptor-mediated pathways and initiate cellular DNA synthesis.
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A single intravenous injection of lead nitrate (LN) to rats induces liver cell proliferation without causing cell necrosis (direct hyperplasia). We suggested that liver cell proliferation in this model may be triggered by the induction of liver tumor necrosis factor alpha (TNF-alpha). Because administration of TNF-alpha in vivo has been shown to induce proliferation of both parenchymal and nonparenchymal cells of the liver, we analyzed the temporal sequences of DNA synthesis in both cell populations following LN and recombinant TNF-alpha treatment by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. The patterns of cell proliferation induced by these agents were further compared with those induced by a single dose of nafenopin (NAF), a direct mitogen which does not induce liver TNF-alpha messenger RNA (mRNA). In male Wistar rats given a single dose of LN (100 micromol/kg), BrdU incorporation of hepatocytes and nonparenchymal cells (Kupffer cells, endothelial cells and periportal nondescript cells) became evident 12 hours after the treatment. The labeling of all cell types reached a peak after 36 hours and declined thereafter. Rats given a single intravenous injection of human recombinant TNF-alpha (46 microg/rat) showed an increase of BrdU labeling in nonparenchymal cells after 24 hours, whereas the labeling of hepatocytes became evident at 36 hours. A single intragastric administration of NAF resulted in a rapid increase in the number of labeled hepatocytes with no substantial labeling of nonparenchymal cells. These results add further support to the notion that LN-induced liver cell proliferation is mediated by TNF-alpha, and suggest that different cell populations are involved in the initial proliferative response of the liver to mitogens, depending on the capacity of the mitogens to stimulate TNF-alpha production.
We recently suggested that peroxisome proliferators (PP)-induced hepatocyte DNA synthesis may be mediated by a specific peroxisome proliferator activated receptor (PPAR). Heterodimers of the PPAR with the retinoid nuclear receptor, RXR, activate transcription after binding to DR1 response elements of the target genes. DR1 elements are also activated by RXR homodimers formed in the presence of 9-cis retinoic acid (9 cis RA) suggesting that PP and 9 cis RA might regulate an overlapping set of target genes. The present study was therefore designed to test whether 9-cis RA stimulates hepatocyte DNA synthesis. Male Wistar rats were given a single intragastric dose of 9-cis RA (10-100 mg/Kg) or all trans retinoic acid (RA)(200 mg/Kg and 100 mg/Kg), and levels of hepatocyte DNA synthesis after 24 hours were determined by BrdU immunohistochemistry. Effects of 9-cis RA and RA(10(-9)-10(-5)M) on hepatocyte DNA synthesis in primary culture were also examined. Over 10 fold increases in the levels of BrdU incorporation were noted 24 hours after a single dose of 9 cis RA at a dose of 60 and 100 mg/Kg. RA at a dose of 200 mg/Kg induced a 5-6 fold increases in BrdU labeling, while a dose of 100 mg/Kg had no significant effects. Since the RA effect only occurs at higher doses, it may be only after conversion to 9-cis RA. In primary culture of hepatocytes, neither 9-cis RA nor RA with or without EGF had stimulatory effects on hepatocyte DNA synthesis. This is the first report to demonstrate a potent stimulatory effect of 9-cis RA on DNA synthesis of rat hepatocytes in vivo. It is suggested that 9-cis RA exerts this effect through receptor mediated mechanisms similar to PP, both activating genes that regulate hepatocyte proliferation.
We examined possible roles of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) in lung morphogenesis. By polymerase chain reaction, transcripts for both KGF and its receptor were detected early (rat gestational days 16 and 14, respectively) and their abundance increased during lung morphogenesis. To evaluate possible role of KGF in lung morphogenesis, day 14 lung explants were cultured in Dulbecco's modified Eagle medium + 10% fetal calf serum for 1 to 4 days in the presence (5-50 ng/ml) or absence of KGF (control). KGF (at 25 and 50 ng/ml) induced a marked reduction in the number of terminal branches and destination of the distal epithelium into cyst-like structures. These effects of exogenous KGF were progressively diminished by increasing concentrations of anti-KGF (2-16 micrograms/ml). Electron microscopic examination revealed that the epithelial cells of the cystic structures contained lamellar bodies, and were therefore type II cells and/or their progenitors. Northern blot analysis showed higher expression of surfactant protein C (SP-C) mRNA (a marker for alveolar epithelial type II cells) in KGF-treated fetal lungs. In situ hybridization of the KGF-treated lungs revealed that the SP-C mRNA-expressing cells were arranged distally in the form of linear arrays, a pattern distinctly different from that in control lungs. Acidic fibroblast growth factor, which also binds KGF receptors, in the presence of heparin mimicked the effect of KGF on branching. Transforming growth factor-beta(1) (TGF-beta 1) inhibited branching of fetal lungs in culture, and this effect dominated over that induced by KGF. Blocking of endogenous HGF with antibodies or addition of HGF to cultures of fetal lung explants had no significant effect on branching or growth. In conclusion, KGF markedly influences branching, and epithelial growth, differentiation, and patterning during lung morphogenesis.
The carcinogenic process in the liver is a multistep process, characterised by an altered ratio between cell proliferation and cell death. In the last few years, we have undertaken studies aimed at determining the possible differences exhibited by two different types of cell proliferation, namely compensatory regeneration and direct hyperplasia at a molecular and cellular level. These two types of proliferative stimuli appear to play different roles in liver carcinogenesis. The scope of this article is to summarise the present knowledge about the differences in the expression of genes involved in the entry of liver cells into cell cycle, between liver regeneration following cell loss and/or cell death and direct hyperplasia induced by primary mitogens.
In compensatory hyperplasia after partial hepatectomy or liver cell injury, hepatocyte proliferation is triggered by coordinated actions of growth factor such as hepatocyte growth factor and transforming growth factor-alpha and -beta. Initiation of hepatocyte DNA synthesis is preceded by the activation of the set of early growth response genes mediated by enhanced nuclear factor-kappa B binding to DNA. Using an experimental model to induce hepatocyte DNA synthesis in vivo by a single dose of a peroxisome proliferator, which does not induce liver cell necrosis (direct hyperplasia), we investigated whether peroxisome proliferator-induced hepatocyte proliferation involved an induction of known growth factors, an activation of early growth response genes, and nuclear factor-kappa B. A single intragastric administration of 250 mg/kg BR931 (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide) to male wistar rats induced a wave of hepatocyte DNA synthesis starting after 12 hours and peaking at approximately 24 to 36 hours. The response was dose dependent. The treatment also induced the expression of the mRNA for the peroxisomal bifunctional enzyme, one of the peroxisome-related fatty acid beta-oxidation enzymes. Pretreatment of rats with dexamethasone (2 mg/kg) inhibited both hepatocyte DNA synthesis and the induction of the peroxisomal bifunctional enzyme gene. Northern blot analyses of liver RNA during a period preceding the onset of DNA synthesis revealed no induction of hepatocyte growth factor, transforming growth factor-alpha, or tumor necrosis factor-alpha mRNAs. No induction of early growth response genes, liver regeneration factor-1, or c-myc was detected. Furthermore, gel mobility shift assays showed no enhanced nuclear factor-kappa B binding to its DNA consensus sequence after BR931 treatment, whereas control studies demonstrated a distinct increase in binding after partial hepatectomy or lead nitrate treatment. The results suggest that peroxisome-proliferator-induced hepatocyte proliferation may be triggered by signal transduction pathways different from those after partial hepatectomy and that the binding of peroxisome proliferators to their nuclear receptors may play a role in stimulation of DNA synthesis and peroxisome proliferation.
Hepatocyte growth factor (HGF) and its receptor, the product of c-MET proto-oncogene, are highly expressed in both fetal and adult lung, though their physiologic functions in the lung are largely unknown. In the present study, we examined whether alveolar type II cells in the lung are the target of HGF and whether HGF has any effects on growth of these cells. The alveolar epithelial type II cells were isolated from the lungs of adult male Sprague-Dawley rats by elastase digestion, and the cells were used to determine whether they express HGF and c-MET mRNAs and whether recombinant HGF has any effect on their DNA synthesis in primary culture. The effects were further compared with those induced by epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), transforming growth factor-alpha (TGF-alpha), and transforming growth factor-beta 1 (TGF-beta 1). Northern blot analysis and in situ hybridization revealed that type II cells express c-MET mRNA but not HGF mRNA. HGF stimulated [3H]thymidine incorporation into type II cells in primary cultures. An increase was also seen in labeling index as determined by nuclear immunostaining of bromodeoxyuridine-incorporated DNA. While aFGF (200 ng/ml) exerted an effect comparable to HGF (25 ng/ml) on DNA synthesis in type II cells, EGF (20 ng/ml) and TGF-alpha (100 ng/ml) had lesser effects. TGF-beta 1, a potent inhibitor of epithelial cell proliferation, at 0.25 to 2 ng/ml, did not inhibit HGF-induced [3H]thymidine incorporation into type II cells. The results indicate that HGF exerts its effects on type II cells as a potent mitogen by a paracrine mode of action.
BACKGROUND: Proteins in human lung lavage were analyzed to identify cell-specific markers for potential use in the study of the biology and pathology of pulmonary cells. EXPERIMENTAL DESIGN: Proteins associated with pulmonary surfactant were used to raise mAb. An Ab to a 130-kDa protein (MCp130) was reactive with ciliated and mesothelial cells. Expression of this Ag in normal organs, developing lung, and tumors was investigated. RESULTS: By Western blotting, the Ab stained a protein of about 130 kDa. In formalin-fixed, paraffin-embedded tissues from adult human and rat organs, the Ab specifically stained the luminal/apical surfaces of pulmonary and nonpulmonary ciliated and mesothelial cells. Staining of fetal airway cells was independent of ciliation. Airway cell staining was detectable in human fetal lungs at 12 weeks of gestation and at Day 18 of gestation in fetal rat. The Ab reacted with human and rat fetal mesothelial cells at the gestational ages of 15 weeks and 17 days, respectively. It also stained ciliated cells in endosalpinx and endometrium. Human epithelial mesotheliomas and ovarian and endometrial carcinomas stained selectively, whereas other pulmonary tumors and tumors of other organs did not react with the Ab. CONCLUSIONS: This 130-kDa mesothelial and ciliated cell plasma membrane protein appears in developing lung at an earlier age than secretory proteins. The marker is of potential use in the study of development of the different cell lineages in the lung and female reproductive tract. The Ab is expected to be useful in the diagnosis of epithelial mesotheliomas and ovarian/endometrial carcinomas, because it selectively stains these tumors and is reactive with formalin-fixed, paraffin-embedded tissues.
We have recently demonstrated that a single injection of the mitogen lead nitrate to rats induced a rapid increase of tumor necrosis factor-alpha (TNF-alpha) mRNA in the liver and suggested that this cytokine may be involved in triggering hepatocyte proliferation in this model of direct hyperplasia. In this study, we examined whether a similar induction of liver TNF-alpha mRNA could be observed preceding the onset of hepatocyte proliferation induced by ethylene dibromide, another hepatocyte mitogen. In addition, we used dexamethasone, a well known inhibitor of TNF-alpha production, to determine whether its administration could suppress hepatocyte proliferation induced by lead nitrate and ethylene dibromide. A single intragastric administration of ethylene dibromide (100 mg/kg) to male Wistar rats enhanced liver TNF-alpha mRNA after 4 and 7 hours, which then returned to control levels by 24 hours. TNF-alpha mRNA was detectable only in a nonparenchymal cell fraction of the liver. Pretreatment of rats with a single dose of dexamethasone (2 mg/kg) 60 minutes before lead nitrate (100 mumol/kg) or ethylene dibromide completely abolished the increased levels of liver TNF-alpha mRNA induced by these agents. Inhibition by dexamethasone of TNF-alpha mRNA was associated with an inhibition of liver cell proliferation induced by these mitogens, as measured by [3H]thymidine incorporation into hepatic DNA, mitotic index, and DNA content. These results further support the hypothesis that TNF-alpha may be involved in triggering hepatocyte proliferation induced by primary mitogens.
BACKGROUND: A single intravenous injection of lead nitrate to rats induces a synchronized wave of hepatocyte proliferation without accompanying liver cell necrosis. However, the mechanism of the mitogenic effect of lead nitrate is not known, and whether hepatocyte growth factor (HGF), transforming growth factor-alpha (TGF-alpha), and transforming growth factor-beta 1 (TGF-beta 1) play any role in it have not been investigated. These growth factors have indeed been shown to provide either positive or negative stimuli for liver cell regeneration after partial hepatectomy or liver cell necrosis. Moreover, there are reports showing that administration of non-necrogenic doses of tumor necrosis factor-alpha (TNF-alpha) to rats lead to an enhanced proliferation of hepatocytes and liver nonparenchymal cells. Lead is known to sensitize animals to lethal effects of bacterial lipopolysaccharides (LPS), suggesting that lead nitrate may modify the production of TNF-alpha in response to endogenous LPS of intestinal origin. An enhanced production of TNF-alpha could therefore be involved in the mitogenic action of lead nitrate. EXPERIMENTAL DESIGN: We investigated first whether a single intravenous dose of lead nitrate (100 mumole/kg) to rats modifies the production of HGF, TGF-alpha, and TGF-beta 1, by examining the steady-state level of their mRNA in the liver by Northern blot analyses. The response of rats given lead nitrate to various doses of LPS was next evaluated to determine whether lead-treated rats have an enhanced sensitivity to LPS. Finally, the level of TNF-alpha mRNA was examined in the liver of rats at various time periods after a single injection of lead nitrate. RESULTS: No changes were observed in the liver levels of mRNAs for HGF, TGF-alpha, and TGF-beta 1 at various time intervals after a single injection of lead nitrate. All rats given only single injections of LPS up to 100 micrograms survived. However, lead nitrate-treated rats tolerated LPS at dosages of only 6 micrograms. The liver of control rats showed a single 1.6 kb TNF-alpha transcript, whereas 1.8-kb transcripts were seen at 1 hour after lead nitrate injection, and persisted for 12 hours. The 1.8 kb TNF-alpha transcript was also present in the spleen of control rats, and its expression was enhanced in lead nitrate-treated rats. CONCLUSIONS: Stimulation of hepatocyte proliferation induced by lead nitrate was not accompanied by changes in liver levels of HGF, TGF-alpha, or TGF-beta 1 mRNA. Lead nitrate, however, enhanced expression of TNF-alpha at a time preceding the onset of hepatocyte DNA synthesis, indicating that TNF-alpha may trigger the lead nitrate-induced proliferation of hepatocytes.
A mouse monoclonal antibody to a human lung lavage protein was raised using proteins, with the potential ability to bind surfactant, as the immunogen. The proteins were isolated from cadaver lung lavage. The antibody was tested for its reactivity with lung and other organs. It reacted with type I pneumocytes and some of the nonciliated cells in the surface epithelium of distal bronchioles. Staining was also seen in the cells surrounding the glandular structures, superficial keratinocytes of the skin, endothelium, and nerve sheath cells. With the exception of bronchiolar cells, the stained cells have a squamous morphology, and this protein may serve as a marker or determinant of this characteristic of cells. In pathologic lungs some of the cells in air spaces with "bronchiolarization" of the epithelium exhibited staining for the protein. It could not be ascertained whether the stained cuboidal cells were reactive type II pneumocytes or distal bronchiolar cells. The intraalveolar material in pulmonary alveolar proteinosis did not show remarkable staining for the protein. Even though the protein is not unique to type I pneumocytes, it may serve as a marker for these cells in the study of their development and biology.
Human Clara cell 10-kDa protein has been suggested to be a counterpart of rabbit uteroglobin, an immunomodulatory and antiinflammatory secretory protein. Since this human protein is not readily available in substantial quantity for detailed characterization of its biochemical, biological, and pharmacological properties, we sought to express it in Escherichia coli in order to study its structure-function relationship. However, bacterial overproduction of homodimeric proteins with interchain disulfide bonds, such as Clara cell 10-kDa protein, was thought to be impossible until we achieved expression of native uteroglobin (Miele, L., Cordella-Miele, E., and Mukherjee, A.B. (1990) J. Biol. Chem. 265, 6427-6435). Here, we report high level production of recombinant native dimeric human Clara cell 10-kDa protein in E. coli and its characterization. Recombinant human Clara cell 10-kDa protein forms its disulfide bonds within the bacterial cytoplasm. The purified protein possesses two of the most important activities characteristic of uteroglobin: (i) it is an excellent substrate of transglutaminase, and (ii) it is a potent inhibitor of porcine pancreatic and, more importantly, human synovial phospholipase A2. These results demonstrate that human Clara cell 10-kDa protein and rabbit uteroglobin have very similar biochemical properties. Our data suggest that this protein may possess immunomodulatory and antiinflammatory activities of potential physiological and pharmacological importance.
A recent study from our laboratory demonstrated that cyclosporine (CsA), a prototype immunosuppressant, enhanced the growth of carcinogen-induced enzyme altered foci in rat liver, suggesting that CsA may stimulate development of hepatocellular carcinomas. In the present study, we examined (i) whether CsA accelerates development of hepatocellular carcinomas in experimental animals, (ii) whether CsA stimulates the proliferation of resting hepatocyte in vivo and (iii) whether CsA modulates the production of growth factors implicated in liver cell growth, hepatocyte growth factor (HGF), transforming growth factor alpha (TGF alpha) and transforming growth factor beta 1 (TGF beta 1). Foci of hepatocytes, positive for glutathione S-transferase placental form were induced in male F344 rats by a single dose of diethylnitrosamine followed by 7 weeks promotion by a choline-deficient diet. The animals were then divided in two groups, and subsequent development of hepatocellular carcinomas was compared in rats fed a basal diet or a basal diet containing 0.015% CsA. Development of hepatocellular carcinoma was accelerated in the rats maintained on a CsA diet. Feeding a CsA diet as the sole treatment, for 2, 4 and 10 weeks induced significant increases in liver weight, and resulted also in an enhanced incorporation by hepatocytes of 5-bromo-2-deoxy-uridine. Serum levels of glutamate-oxaloacetate transferase, glutamate-pyruvate transferase and lactic dehydrogenase were not altered by feeding a CsA diet. Northern Blot analyses of the expression of HGF, TGF alpha and TGF beta 1 mRNAs in the liver showed similar patterns of expression between rats fed a basal diet and a CsA diet. The levels of HGF mRNA were not altered in the lungs and kidneys of rats fed a CsA diet. These results indicate that CsA stimulates rat liver cell proliferation in vivo without inducing liver cell necrosis, and that this effect may contribute to accelerated development of hepatocellular carcinomas in rats fed a CsA diet. As previously observed with BR 931, a hypolipidemic peroxisome proliferator, stimulation of liver cell growth by CsA did not entail changes in the production of HGF, TGF alpha or TGF beta 1.