Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors.

BACKGROUND AND OBJECTIVE: Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts. MATERIAL AND METHODS: Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription-polymerase chain reaction analysis. RESULTS: We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens. CONCLUSION: Taken together, our data highlight the importance of arecolineinduced epithelial changes in the pathogenesis of oral submucous fibrosis.

Transglutaminase-2 regulation by arecoline in gingival fibroblasts.

Transglutaminase-2 (TGM-2) stabilizes extracellular matrix (ECM) proteins by cross-linking and has been implicated in several fibrotic disorders. Arecoline present in betel quid has been proposed as one of the causative factors for oral submucous fibrosis (OSMF). Hence, we hypothesize that arecoline may regulate TGM-2 and may have a role in the pathogenesis of OSMF. The expression of TGM-2 was studied in OSMF tissues by real-time RT-PCR analysis, and significant overexpression was observed in most OSMF tissues (P=0.0112) compared with normal tissues. Arecoline induced TGM-2 mRNA and protein expression as well as TGM-2 activity in human gingival fibroblast cells. The addition of methocramine hemihydrate (M-2 muscarinic acetylcholine receptor selective antagonist) or 8'-bromo-cAMP abolished arecoline-mediated TGM-2 induction, suggesting a role for M-2 muscarinic acid receptor and a repressor role for cAMP. Our study provides evidence for TGM-2 overexpression in OSMF and its regulation by arecoline in oral fibroblasts.

PKC activity and protein phosphorylation in regulation of sIg mediated B cell activation.

The inhibitory and stimulatory elements of cellular signalling associated with activation of protein kinase C (PKC) in murine B lymphocytes were investigated by employing two PKC activators with opposing effects on cell proliferation. Being an inhibitor of anti-Ig mediated proliferation, the phorbol ester PDBU induced a more substantial translocation of cytosolic PKC activity than the alkaloid PKC activator indolactam, which enhances anti-Ig mediated B cell proliferation. PDBU and indolactam were equally effective kinase activators, as determined by 32P incorporation of the substrate proteins. Concentrations of indolactam which induced an inhibition of anti-Ig mediated B cell proliferation also induced a precipitous decline in detergent soluble cellular PKC activity, which was comparable with 1 microM PDBU. The induced phosphoprotein patterns were similar, with an exception of the nuclear envelope protein lamin B, which was prominently phosphorylated by PDBU but not by stimulatory concentrations of indolactam. The enhanced phosphorylation of lamin B was associated with cellular growth arrest: inhibitory concentrations of indolactam induced the phosphorylation of lamin B equal to PDBU, whereas an increased phosphorylation of lamin B was never observed upon stimulation with anti-Ig. Together, inhibition of anti-Ig mediated B cell proliferation was related to down-regulation of cytoplasmic PKC and induction of nuclear PKC-dependent phosphorylation.

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.

Studies on hormonal modulation of asparagine-linked glycoprotein biosynthesis in explant cultures of rat mammary gland.

Glucosidase I has been purified to homogeneity and polyclonal antibodies against the enzyme have been prepared. The anti-glucosidase I antibodies recognized a single band of 85 kDa on western blot at a dilution as high as 1:2000 and also inhibited the enzyme activity, suggesting the specificity of the antibodies. Con A-Sepharose binding experiment indicates that this enzyme itself is a high mannose type N-linked glycoprotein. The increase in the electrophoretic mobility of 85 kDa band following digestion with endoglycosidase H and F strengthened this observation. The presence of any O-linked sugar attached covalently to glucosidase I could not be detected by binding assays with O-linkage specific biotinylated lectins. The studies on developmental regulation suggest that the synthesis of glucosidase I is modulated with the ontogeny of the gland. Lactogenic hormones, viz. insulin, hydrocortisone and prolactin, appeared to regulate the synthesis of glucosidase I. The possible role of these hormones in the overall regulation of protein N-glycosylation has been discussed.

Developmental regulation of glucosidase I, an enzyme involved in the processing of asparagine-linked glycoproteins in rat mammary gland.

Glucosidase I involved in the processing of N-linked glycoproteins was purified to homogeneity from the lactating rat mammary gland. The purified enzyme exhibited a single band at 85 kDa on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Polyclonal antibodies raised against the enzyme recognized a similar band on Western blots and also inhibited the enzyme activity. The enzyme levels gradually increased until the midlactation stage and thereafter declined sharply during the period of postlactation. A similar profile of the levels of immunoreactive glucosidase I was observed. These findings suggest that the accumulation of glucosidase I is modulated as a function of gland ontogeny. The results on hormonal regulation of glucosidase I indicate that the synthesis of the enzyme is stimulated by a combination of insulin, hydrocortisone, and prolactin; additionally, epidermal growth factor may play a role in this regulation. The above observation was substantiated by immunoprecipitation of [35S]methionine-labeled microsomal extracts with anti-glucosidase I antibodies. The immunoprecipitation of soluble extracts from [35S]methionine-labeled tissue with anti-rat alpha-lactalbumin antibodies indicates that these hormones not only stimulate the synthesis of alpha-lactalbumin but also play an important role in its glycosylation.

Prostaglandin F2 alpha receptors on enzyme-dissociated pig luteal cells throughout the estrous cycle.

A deficiency of luteal cell prostaglandin F2 alpha (PGF2 alpha) receptors might help explain the well documented refractoriness of pig corpora lutea to the luteolytic effects of PGF2 alpha administered in vivo before day 12 of the estrous cycle. Accordingly, experiments were conducted to measure the levels of [3H] PGF2 alpha-binding sites/receptors on collagenase-dispersed pig luteal cells taken at different stages of the estrous cycle. Pig corpora lutea were obtained surgically at various stages of the estrous cycle and dissociated with collagenase in medium 199. Dissociated mixed luteal cells (approximately 5-15 x 10(4) large luteal cells/tube) were assayed for [3H]PGF2 alpha-binding activity by saturation (Scatchard) analysis. In preliminary experiments it was determined that PGF2 alpha binding was maximal after incubation for 45 min at 30 C in assay buffer of pH 5.75. Additionally, it was determined that [3H]PGF2 alpha binding was displaceable by PGF2 alpha = PGD2 greater than PGE2 greater than 13,14-dihydro-15-keto-PGF2 alpha. Other eicosanoids did not inhibit [3H]PGF2 alpha binding. Two distinct classes of binding sites (high affinity Kd = 19-64 nM; low affinity Kd = 262-3103 nM) were observed at all stages of the estrous cycle. From studies using enriched (by elutriation) large (greater than 30 microns) and small (10-20 microns) luteal cells it appeared that the high affinity binding site was largely confined to large luteal cells, whereas the low affinity binding site was found on both large and small luteal cells. The concentrations (number per large luteal cell) of high affinity PGF2 alpha-binding sites of mixed (unelutriated) luteal cell preparations was low on days 6-8 (0.6 x 10(6) sites/cell) and increased gradually up to 1.4 x 10(6) sites/cell on day 12. The concentrations of binding sites were increased approximately 3-fold on day 13 (4.6 x 10(6) sites/cell; P less than 0.05 vs days 6-12) and remained elevated on days 14 and 16-17 (approximately 3 x 10(6) sites/cell). In summary, these results indicate the existence of a high affinity PGF2 alpha-binding site in pig (large) luteal cells, which is probably the luteal PGF2 alpha receptor. The numbers of these putative PGF2 alpha receptors are low during the early luteal phase (before day 12), but increase thereafter (days 13, 14, and 16-17). This may provide one explanation for the observed refractoriness in vivo of pig corpora lutea to PGF2 alpha before, and increased sensitivity to PGF2 alpha after, day 12 of the estrous cycle.

Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells.

A cryostorage procedure was developed to provide ovine luteal cells throughout the period of seasonal anestrus. Corpora lutea obtained from midluteal phase, superovulated ewes were dispersed enzymatically. Some dispersed cells were fractionated into subpopulations by elutriation. Dimethylsulfoxide (7.5% final concentration) in Hanks' buffered saline was added to cells at 4 degrees C, and dispersed cell preparations were frozen in a programmable cell freezer and stored at -196 degrees C. After recovery from cryopreservation, cell viability and prostaglandin F2 alpha (PGF2 alpha) binding characteristics of thawed cells were not different from those of corresponding fresh cells. Additionally, thawed cells retained the capacity to attach to culture dishes and retained responsiveness of progesterone secretion to prostaglandin E2 (PGE2) and ovine luteinizing hormone (LH), although rates of progesterone secretion were attenuated in thawed compared with fresh cells. The cryopreservation procedure will prove useful to relieve constraints in utilization of ovine luteal cells arising from reproductive seasonality in sheep. Cells retrieved from cryostorage were evaluated by studying PGF2 alpha binding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF2 alpha) of PGF2 alpha binding to unfractionated cells, we detected a single class of high affinity binding sites (Kd = 17.4 +/- 2.3 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF2 alpha and increasing amounts of unlabeled PGF2 alpha) and unfractionated cells, we detected additional binding sites of lower affinity (Kd = 409 +/- 166 nM) as well as the nonspecific binding component. Small luteal cells obtained by elutriation, which were essentially devoid of large cell contamination, had only low affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural requirements for prostaglandin analog interaction with the ovine corpus luteum prostaglandin F2 alpha receptor. Implications for development of a photoaffinity probe.

The capacity of structurally modified analogs of prostaglandin F2 alpha (PGF2 alpha) to inhibit binding of [3H]PGF2 alpha to receptors on ovine luteal cells was evaluated by radioreceptor assay using dispersed, viable, ovine luteal cells. Binding assays were conducted at pH 5.75, since binding to both high (Kd 17.4 +/- 2.3 nM) and low (Kd 409 +/- 166 nM) affinity sites was enhanced markedly at reduced pH. The capability to compete with [3H]PGF2 alpha for binding was evaluated for different prostaglandin analogs having modifications in the C-8 "upper" side-chain, in the cyclopentane ring, or in the C-12 "lower" side-chain. Prostaglandin J2 was a surprisingly potent competitor for binding to the PGF2 alpha receptor. Several phenyl-substituted analogs exhibited receptor-binding potency greater than or equal to native PGF2 alpha, while most other analogs had reduced capacity to compete with native PGF2 alpha for binding. Several 17-azidophenol PGF2 alpha analogs were synthesized and tested, but analogs having hydroxyl groups on the aryl ring had low affinity for receptors. However, 17-(4-azidophenyl)-18,19,20-trinor-PGF2 alpha as well as 17-(3-iodo-4-azidophenyl)-18,19,20-trinor-PGF2 alpha exhibited binding affinities that were approximately 10% of native PGF2 alpha, and the radioiodinated analogs of PGF2 alpha may be useful as probes of the PGF2 alpha receptor.

Ontogeny of alpha-foetoprotein in human foetal brain.

Alpha-foetoprotein (AFP) was found to be present in human foetal brain during early embryonic life. Antigenically and electrophoretically foetal brain AFP is similar to that found in human foetal serum. Like serum AFP it did not bind oestradiol. Developmental profile of the proteins in foetal brain was distinct from that observed in foetal serum, amniotic fluid and maternal serum compartments. Peak levels of brain AFP were obtained in foetuses in the 20th week of gestation. These levels decreased rapidly and no AFP could be detected in foetal brains derived from the third trimester of pregnancy. The origin of AFP and its possible role in foetal brain development is discussed.