Aging is associated with increased proliferation and decreased apoptosis in the colonic mucosa.

Although the incidence of colon cancer increases with advancing age, reasons for this increase are not fully understood. Earlier studies have demonstrated that in Fischer-344 rats, aging is associated with increased crypt cell production in the colon, an event considered to be central to the initiation of carcinogenesis. Apoptosis also plays a critical role in the development and progression of colon cancer. Therefore, we have examined the age-related changes in proliferation and apoptosis in the colonic mucosa of 4-5, 12-14, and 22-24 month-old Fischer-344 rats. We have observed that proliferative activity in the colon, as assessed by proliferating cell nuclear antigen immunoreactivity, is higher (50-80%) in 12-14 and 22-24 month-old rats than in their 4-6 month-old counterparts. In contrast, the number of apoptotic cells, (as determined by TdT-mediated dUTP nick-end labeling assay) in the colonic mucosa of 12-14 and 22-24 month-old rats are considerably lower (50-60%) than in 4-6 month-old animals. These changes are accompanied by a concomitant reduction (75%) in pro-apoptotic Bak and stimulation (200%) of anti-apoptotic Bcl-xL levels. Since activation of caspases is associated with initiation and maintenance of apoptosis, we also analyzed the levels of pro and active forms of caspase-3, 8 and 9. The levels of active forms of caspase-3, 8 and 9 are found to be considerably (60-80%) lower in the colonic mucosa of 22-24 month-old rats, compared to their younger counterparts. This is accompanied by decreased cleavage of poly(ADP-ribose) polymerase, a substrate for caspases. In conclusion, our data show that aging enhances proliferation, but attenuates apoptosis in the colonic mucosa. These changes may partly be responsible for the age-related rise in colorectal cancer.

Inhibition of type I procollagen synthesis by damaged collagen in photoaged skin and by collagenase-degraded collagen in vitro.

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

Aging alters gastric mucosal responses to epidermal growth factor and transforming growth factor-alpha.

Administration of pharmacological doses of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) in young rats stimulates gastric mucosal proliferation, but, in aged rats, the same treatment inhibits proliferation. This may be due to enhanced ligand-induced internalization of EGF receptor (EGFR). In support of this, we demonstrated that although a single injection of EGF (10 microg/kg) or TGF-alpha (5 microg/kg) in young (4-6 mo old) rats greatly increased membrane-associated EGFR tyrosine kinase activity, the same treatment slightly inhibited the enzyme activity in aged (24 mo old) rats. This treatment also produced a greater abundance of punctate cytoplasmic EGFR staining in gastric epithelium of aged rats, consistent with EGFR internalization. In vitro analyses demonstrated that exposure of isolated gastric mucosal cells from aged but not young rats to 100 pM TGF-alpha resulted in marked increases in intracellular EGFR tyrosine kinase activity and that induction of EGFR tyrosine kinase activity in mucosal membranes from aged rats occurred at doses 1,000-fold less than those required in young rats. Our data suggest that aging enhances sensitivity of the gastric mucosa to EGFR ligands. This may partly explain EGFR-mediated inhibition of gastric mucosal proliferation in aged rats.

Induction of EGFR tyrosine kinase in the gastric mucosa of diabetic rats.

Diabetes mellitus is associated with spontaneous gastric mucosal injury and enhanced susceptibility of the mucosa to damaging agents. Little information is available about the biochemical changes that occur in the gastric mucosa of diabetes mellitus. Evidence is accumulating that tyrosine kinases, particularly the EGF-receptor (EGFR), are involved in regulating a variety of structural and functional properties of the gastric mucosa. The primary objectives of this investigation were to determine whether diabetes induces morphological changes in the gastric mucosa, and if so, whether these changes are associated with alterations in EGFR tyrosine kinase. Diabetes-induced changes in gastric mucosal morphology were also examined. Diabetes was induced in 3- to 4-month-old male Fischer-344 rats by streptozotocin (STZ; 45 mg/kg; i.v.). Four weeks after induction of diabetes mellitus, the gastric mucosa of overnight-fasted rats was found to be slightly atrophic. A reduction in gastric mucosal thickness with deposition of fibrous tissue above the muscularis layer was observed in the stomach of overnight-fasted diabetic rats. These changes were associated with a marked stimulation in tyrosine kinase activity and protein expression of EGFR. The relative concentrations of several precursor forms of TGF-alpha in both membrane and cytosolic fractions from the gastric mucosa of overnight-fasted diabetic rats were also found to be significantly above the corresponding controls. This suggests that endogenous TGF-alpha may play a critical role in regulating mucosal EGFR tyrosine kinase through a juxtacrine/paracrine mechanism.

Differential expression of EGFR during early reparative phase of the gastric mucosa between young and aged rats.

Aging is associated with decreased reparative ability of the gastric mucosa. Our recent data suggest a role for epidermal growth factor receptor (EGFR) in the mucosal reparative processes. Thus we examined changes in EGFR tyrosine kinase activity as well as expression and subcellular localization of EGFR and its ligand transforming growth factor-alpha (TGF-alpha) in the gastric mucosa of young (4-mo-old) and aged (24-mo-old) Fischer 344 male rats during the early reparative phase after acute injury induced by 2 M NaCl. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. Expansion of the neck region and initiation of foveolar cell migration could be seen within 45 min of injury in young rats but not until 90 min in aged rats. In young rats mucosal EGFR tyrosine kinase activity increased at 45 min after injury and subsequently fell to basal levels. Mucosal EGFR mRNA increased throughout the reparative phase as did content of the EGFR ligand TGF-alpha. In contrast, although the basal tyrosine kinase activity and levels of EGFR mRNA and TGF-alpha were elevated in the gastric mucosa of aged rats, injury did not cause increases in these parameters. Immunofluorescent localization suggests that internalization and/or degradation of EGFR may be higher in aged than in young rats. We suggest that diminished induction of EGFR tyrosine kinase activity and increased EGFR internalization after injury may in part be responsible for the age-related decrease in the reparative capacity of the gastric mucosa.

Tracheobronchial histopathology in habitual smokers of cocaine, marijuana, and/or tobacco.

BACKGROUND: Marijuana and alkaloidal cocaine ("crack") are the two most commonly smoked substances in the United States after tobacco. While regular tobacco smoking has been found to be associated with extensive microscopic alterations in bronchial mucosa, little information is available concerning the effect of crack cocaine and marijuana on tracheobronchial histopathology. STUDY OBJECTIVE: To determine the relative impact of smoked substances (cocaine, marijuana, and tobacco) alone and in combination on the histopathology of the tracheobronchial mucosa and to assess whether the effects of habitual smoking of two or more substances (cocaine, marijuana, and/or tobacco) are additive. DESIGN: Observational cohort study. SUBJECTS: Fifty-three nonsmoking control subjects (NS), 14 current, habitual smokers of crack cocaine only (CS), 40 current, regular smokers of marijuana only (MS), 31 regular smokers of tobacco only (TS), 16 current smokers of both cocaine and marijuana (CMS), 12 current smokers of both cocaine and tobacco (CTS), 44 current smokers of both marijuana and tobacco (MTS), and 31 current smokers of cocaine, marijuana, and tobacco (CMTS). METHODS: After preliminary screening evaluation, including a detailed respiratory and general health questionnaire and routine pulmonary function studies, subjects underwent fiberoptic bronchoscopy with endobronchial biopsies of the mucosa of the primary carina and randomly selected secondary or tertiary carinae. Biopsy specimens were processed for light microscopy, stained with hematoxylin-eosin or periodic acid-Schiff, and examined to assess epithelial, basement membrane, and submucosal alterations by one or two pathologists who were masked to the smoking status of the subject. RESULTS: Smokers of cocaine, marijuana, or tobacco alone all exhibited more frequent abnormalities than NS in 10 (CS) or all 11 (MS and TS) of the histopathologic features assessed. For most features, MS and TS showed significantly more frequent alterations than NS (p < or = 0.02), while CS showed significantly more frequent abnormalities than NS in only three features (p<0.05) and nearly significant differences from NS in two additional features (p < or = 0.09). Alterations were noted most frequently in CTS (six features) and MTS (three features), while abnormalities were relatively infrequent in CMS. For 10 features, MTS had more frequent alterations than MS and TS. With a single exception, CMTS did not show more frequent alterations than CTS or MTS. CONCLUSION: Marijuana and tobacco smoking each produces significant bronchial mucosal histopathology and the effects of marijuana and tobacco appear additive. Cocaine appears to lead to fewer significant bronchial mucosal alterations than marijuana or tobacco when smoked alone and does not add to the changes associated with marijuana. When smoked together with tobacco, however, cocaine appears to augment the bronchial injury caused by tobacco smoking.

Increased expression of EGFR in gastric mucosa of aged rats.

Although in Fischer-344 rats aging is found to be associated with increased gastric mucosal proliferative activity, little is known about the intracellular events that regulate this process. The present investigation examines the age-related changes in gastric mucosal tyrosine kinase activity and expression of epidermal growth factor receptor(EGFR) and its structural and functional analog p185c-erbB-2, the protein product of c-erbB-2/c-neu protooncogene. We observed a significantly higher intrinsic tyrosine kinase activity and tyrosine phosphorylation of EGFR and p185c-erbB-2 in the gastric mucosa of 24-mo-old (aged) rats than in that of their 4- or 12-mo-old counterparts. This was associated with increased levels of EGFR protein and steady-state mRNA levels of EGFR and p185c-erbB-2. In addition, we also observed threefold higher steady-state mRNA levels of transforming growth factor-alpha (TGF-alpha; one of the primary ligands of EGFR) in the gastric mucosa of aged rats than in that of 4-mo-old (young) animals. This was accompanied by a fivefold increase in the relative concentration of the 18-kDa precursor form of TGF-alpha in gastric mucosal membranes but not in the cytosol. In conclusion, our data demonstrate that aging is associated with increased tyrosine kinase activity of EGFR and p185c-erbB-2 in the gastric mucosa. Moreover, the observation that aging results in increased accumulation of TGF-alpha in gastric mucosal membranes raises the possibility that the membrane-bound TGF-alpha could partly be responsible for the constitutively active EGFR-induced signaling pathway in the gastric mucosa of aged rats and, in turn, for stimulation of mucosal proliferative activity.

Gastric mucosal injury and repair: effect of aging.

Although the gastric mucosa of healthy adult animals possesses the inherent capacity to promptly repair (often within 24 h) after a minor to moderate injury, aging appears to diminish its reparative capacity. At least two different repair mechanisms are thought to participate in full repair of the damaged gastric mucosa: the initial rapid process of mucosal restitution begins by migration of viable epithelial cells from gastric pits and glands; the subsequent slower process is replacement of lost cells by cell division. Intracellular events that regulate these processes are poorly understood, nor do we know how they may be affected by aging. However, evidence is accumulating which suggests that a number of gastrointestinal hormones/growth factors, most notably EGF and TGF-alpha may play a critical role in regulating gastric mucosal reparative processes. Since EGF and TGF-alpha exert their physiological actions by activating the intrinsic tyrosine kinase (Tyr-k) activity of their common receptor, the EGF-R, studies have been performed to assess the role of EGF-R Tyr-k in regulating mucosal reparative processes during aging. Recent data suggest that the age-related decline in mucosal repair after acute injury could in part be due to decreased activation of EGF-R Tyr-k. In addition, polyamines and prostaglandins are also thought to be involved in gastric mucosal reparative processes. Although the involvement of polyamines in gastric mucosal reparative processes during aging has not yet been studied, decreased mucosal prostaglandin levels in the aged are thought to be a causative factor for the increased susceptibility of the mucosa to injury. These and other relevant matters are discussed in the current review.

Differential responsiveness of proximal and distal colonic mucosa to gastrin.

In vivo and in vitro experiments were performed to examine the responsiveness of the proximal and distal colonic mucosa to the growth-promoting action of gastrin. Infusion (osmotic minipump) of gastrin G-17-I (250 ng/kg/h) for 5 days to 4-month-old male Fischer-344 rats resulted in a significant (90-150%) increase in proliferative activity (as assessed by BrdU or PCNA immunoreactivity) in the distal colonic mucosa. In contrast, gastrin caused no apparent change in proliferative activity in the proximal colon. Because tyrosine kinases (Tyr-ks) are thought to be critically involved in regulating the trophic action of gastrin, responsiveness of isolated colonocytes from both segments of the colon to gastrin (1 x 10(-9) M) was also examined. Exposure of isolated colonocytes from the distal, but not from the proximal, colon to gastrin for 2 min resulted in a significant (73%) stimulation in Tyr-k activity. This was also accompanied by a marked rise in phosphorylation of at least six membrane proteins with M, of 55, 60, 70, 94, and 170 kDa. Tyr-k activity induced by gastrin in colonocytes from the distal colon was inhibited by tyrphostin (3.2 microM) but not by staurosporine (20 nM). In colonocytes from the distal colon, gastrin also stimulated phospholipase C (PLC) activity, which could also be inhibited by tyrphostin, but not by staurosporine. We conclude that mucosa of the distal, but not the proximal, colon responds to the trophic action of gastrin. Tyr-ks are thought to be involved in the regulation of this process.

Inhibition of gastric mucosal regeneration by tyrphostin: evaluation of the role of epidermal growth factor receptor tyrosine kinase.

Although induction of mucosal cell proliferation is a crucial event in gastric mucosal regeneration after injury, intracellular regulatory processes have not been fully elucidated. We hypothesize that tyrosine kinases (Tyr-k)--specifically the enzyme associated with epidermal growth factor receptor (EGF-R)--play an important role in mucosal regeneration. Utilizing tyrphostin--a Tyr-k inhibitor with a greater specificity for EGF-R Tyr-k than for other Tyr-ks--we have examined the role of EGF-R Tyr-k in gastric mucosal regeneration after injury. Gastric mucosal injury in 3-to 4-month-old rats was induced by orogastric administration of 2 mol/L NaCl, whereas the control animals received an equivalent volume of water. The animals were killed 24 hours later. During this 24-hour experimental period (reparative phase), one of the groups was also injected (IP) with tyrphostin-51 (0.65 mg/kg in 30% dimethyl sulfoxide), whereas the control group received the vehicle. In the absence of tyrphostin, the gastric mucosa showed signs of extensive regeneration, whereas in its presence the degree of regeneration was greatly attenuated. These changes were accompanied by parallel alterations in the number of proliferating cell nuclear antigen-immunoreactive cells and the Tyr-k activity of EGF-R. In water-fed control animals, tyrphostin also caused a significant 30% reduction in proliferating cell nuclear antigen-immunoreactive cells. In these animals, the Tyr-k activity of EGF-R was also decreased by 30%. At 24 hours after injury, EGF-R mRNA levels were increased 36-fold over the water-fed controls, and this increase was not significantly affected by tyrphostin. Our current data suggest that activation of EGF-R is an important event in mucosal regeneration.

Azoxymethane enhances ligand-induced activation of EGF receptor tyrosine kinase in the colonic mucosa of rats.

Recent observations suggest that transforming growth factor alpha (TGF-alpha), which binds to the epidermal growth factor (EGF) receptor (EGFR), may induce neoplastic growth of the colonic mucosa through an autocrine mechanism. To assess the functional role of TGF-alpha in colonic carcinogenesis the present investigation examines the changes in TGF-alpha-and EGF-induced activation of intrinsic tyrosine kinase (Tyr-k) activity of EGFR in the colonic mucosa of rats after administration of the colonic carcinogen azoxymethane (AOM; 20 mg/kg body wt). Five days after a single injection of AOM to 4- to 5-month old rats proliferative activity (as assessed by 5-bromo-2'-deoxyuridine immunoreactivity) in the colonic mucosa was increased by approximately 700% over the corresponding saline-injected controls. This was accompanied by: (i) a marked rise in autophosphorylation of a number of mucosal proteins, including one with a M(r) of 170 kDa, a molecular mass that corresponds to EGFR; (ii) a 110-130% increase in basal EGFR Tyr-k activity. Despite this rise in basal EGFR Tyr-k activity, exposure of isolated colonocytes or detergent-solubilized colonic mucosa from AOM-treated animals to either 1 x 10(-8) M TGF-alpha or EGF caused a further 90-160% increase in EGFR Tyr-k activity over the corresponding basal levels. In contrast, bombesin produced no apparent change in EGFR Tyr-k activity. We conclude that increased ligand-induced activation of EGFR Tyr-k may be an important event for development of the hyperproliferative state associated with induction of colorectal neoplasia.

Induction of EGF-receptor tyrosine kinase during early reparative phase of gastric mucosa and effects of aging.

BACKGROUND: Although the gastric mucosa of adult healthy animals possesses a remarkable capacity to promptly repair its mucosal architecture after an acute injury, aging attenuates this process. We hypothesize that certain tyrosine kinases (Tyr-k), specifically the enzyme associated with EGF-receptor (EGF-R), may play a role in this process. The present investigation was undertaken to evaluate the role of this enzyme in the early reparative phase of the gastric mucosa in young and aged rats. EXPERIMENTAL DESIGN: In our initial effort to test the hypothesis, we examined the changes in both total and EGF-R-associated Tyr-k activities in the gastric mucosa of young adult rats (4-months old) during the first 60 minutes after hypertonic saline (2 M NaCl; 1.5 ml/130 g body weight)-induced injury. Because the maximal stimulation (90-100% over the controls) in both total and EGF-R-associated Tyr-k occurred at 30 minutes after injury, we used this time point to perform the next experiment, in which groups of young and aged rats were given (intragastically) 2 M NaCl or water. One of the young and aged groups of rats was also injected (i.p.) with the Tyr-k inhibitor tyrphostin-51 (300 micrograms/kg body weight) 60 minutes before injury. The gastric mucosa was assayed for EGF-R Tyr-k activity and tyrosine phosphorylation and expression of EGF-R, phospholipase C (PLC) activity and relative concentration and tyrosine phosphorylation of PLC-gamma 1, as well as transforming growth factor-alpha (TGF-alpha) levels. RESULTS: Basal EGF-R Tyr-k activity and the extent of tyrosine phosphorylation of EGF-R, as well as PLC activity, were all found to be higher in the gastric mucosa of aged than in young rats. Although 30 minutes after injury, EGF-R Tyr-k activity, tyrosine phosphorylation of EGF-R, and relative abundance of the receptor were all increased in the gastric mucosa of both young and aged rats, the magnitude of stimulation of each of the parameters was found to be considerably lower in aged than in young rats, compared with the corresponding basal levels. A similar phenomenon was also observed for PLC activity and tyrosine phosphorylation of PLC-gamma 1. The relative concentration of mucosal PLC-gamma 1 level was, however, not affected by injury in either young or aged rats. Tyrphostin greatly attenuated the injury-induced increases in the above mentioned parameters in both young and aged rats. In young but not in aged rats, injury caused a significant increase in mucosal TGF-alpha levels. CONCLUSIONS: We conclude that (a) activation of EGF-R Tyr-k is an important event in the early reparative process of the gastric mucosa, and (b) local production of TGF-alpha may play an important role in regulating the activation of EGF-R Tyr-k.

Modulation of adhesive properties of DEAE-dextran with laminin.

Human squamous epithelial cells produce lower amounts of laminin and fibronectin when cultured on DEAE-dextran than when cultured on gelatin-coated polystyrene (Biotechnol. Bioeng., 33:1235). The epithelial cells also spread much more slowly on DEAE-dextran than they do on gelatin-coated polystyrene. To determine if the low level of matrix production by cells grown on DEAE-dextran contributed to the slowness of cell spreading on this substrate, microcarriers made from DEAE-dextran were treated with exogenous laminin (10 micrograms/cm2 of surface area) and then examined for ability to support cell adhesion. Squamous epithelial cells spread as rapidly on the laminin-treated DEAE-dextran as they did on gelatin-coated polystyrene (much more rapidly than on untreated DEAE-dextran). This indicates 1) that laminin can bind to DEAE-dextran in a fashion that is biologically usable by anchorage-dependent cells, and 2) that when laminin is bound to DEAE-dextran, the failure of squamous epithelial cells to rapidly spread is overcome. These data support the hypothesis that failure of the cells to synthesize an intact extracellular matrix on DEAE-dextran is responsible, at least in part, for the slowness with which cells spread on this substrate.

Identification and evaluation of the role of endogenous tyrosine kinases in azoxymethane induction of proliferative processes in the colonic mucosa of rats.

Although tyrosine kinases (Tyr-k) are known to play a role in regulating proliferation of normal, preneoplastic and neoplastic cells, little is known about the identity of different species of Tyr-k involved in this process. Utilizing a non-denaturing polyacrylamide gel electrophoresis system, in which the separated proteins from tissue extracts are assayed directly for Tyr-k, we attempted to identify the species of Tyr-k that may be involved in azoxymethane (AOM) induction of colonic mucosal ornithine decarboxylase (ODC) activity, an enzyme whose activity is known to rise in rapidly proliferating cells. We have observed that 5 days after a single injection of the colonic carcinogen AOM (20 mg/kg body wt) to 3-4-month old rats, a significant 230% rise in colonic mucosal proliferative activity (as evidenced by 5-bromo-2'-deoxyuridine (BrdU) immunoreactivity) was also accompanied by a 550% increase in ODC activity. This was also associated with a marked rise (140-240%) in the relative activity of Tyr-k of three mucosal proteins with MI of 165, 145 and 125 kDa. Since the molecular mass of one of the Tyr-k (165 kDa) corresponded to that of EGF-receptor (EGF-R), this led us to examine the role of EGF-R Tyr-k in AOM induction of colonic mucosal ODC. We observed that a 320% increase in mucosal ODC activity, 5 days after AOM injection, was accompanied by over 200% rise in Tyr-k activity of EGF-R. Daily injection of tyrphostin (300 micrograms/kg body wt.), a Tyr-k inhibitor with a higher specificity for EGF-R Tyr-k, significantly attenuated AOM-induced stimulation of both ODC and Tyr-k activity of EGF-R. Administration of AOM also stimulated the rate of synthesis and secretion of TGF-alpha in isolated colonocytes. In addition, the levels of TGF-alpha and its mRNA in the colonic mucosa were also found to be 100% and 250% higher, respectively, in AOM-treated rats when compared with the controls. We suggest that (a) activation of intrinsic Tyr-k of EGF-R is an important event in AOM induction of colonic mucosal proliferative processes, and (b) this activation is thought to be mediated by TGF-alpha through an autocrine mechanism.

Human skin in organ culture. Elaboration of proteolytic enzymes in the presence and absence of exogenous growth factors.

Proteinase levels were assessed in organ culture fluids from human neonatal foreskin maintained under growth factor-free conditions and in the presence of a combination of growth factors (ie, epidermal growth factor, insulin, hydrocortisone, pituitary extract, and all-trans-retinoic acid). Analysis of culture fluids by gelatin zymography revealed the presence of 92-kd and 72-kd gelatinases. There was a greater amount of 92-kd gelatinase activity in the presence of growth factors whereas the levels of 72-kd gelatinase were similar in growth factor-free and growth factor-containing media. Experiments with keratinocytes and fibroblasts in monolayer culture and with isolated dermal tissue in organ culture indicated that the epithelial component was responsible for most of the 92-kd gelatinase activity whereas fibroblasts were primarily responsible for the 72-kd gelatinase activity. Activation with aminophenyl mercuric acetate, requirement for divalent cations, inhibition with EDTA, and insensitivity to inhibition with phenylmethyl sulfonyl fluoride indicated that both gelatinases were metalloproteinases. In additional studies, culture fluids were examined for the presence of plasminogen activator activity. This was detected in culture fluids from tissues maintained under both conditions but was increased in the growth factor-containing medium. The increased amount seen in the growth factor-containing medium appeared to be due almost entirely to a single factor, ie, all-trans-retinoic acid. In monolayer culture, both keratinocytes and fibroblasts produced plasminogen activator; the level was higher in keratinocyte culture fluids than in culture fluids from fibroblasts.

All-trans retinoic acid (RA) stimulates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to RA while neonatal foreskin responds differently.

Adult human skin from a sun-protected site (hip) and from a sun-exposed site (forearm) was maintained in organ culture for 12 d in the presence of a serum-free, growth factor-free basal medium. Cultures were incubated under conditions optimized for keratinocyte growth (i.e., in 0.15 mM extracellular Ca2+) or for fibroblast growth (i.e., in 1.4 mM extracellular Ca2+). Treatment with all-trans retinoic acid (RA) induced histological changes in the organ-cultured skin under both conditions which were similar to the changes seen in intact skin after topical application. These included expansion of the viable portion of the epidermis and activation of cells in the dermis. In sun-damaged skin samples, which were characterized by destruction of normal connective tissue elements and presence of thick, dark-staining elastotic fibers, a zone of healthy connective tissue could be seen immediately below the dermo-epidermal junction. This zone was more prominent in RA-treated organ cultures than in matched controls. Associated with these histological changes was an increase in overall protein and extracellular matrix synthesis. In concomitant studies, it was found that RA treatment enhanced survival and proliferation of adult keratinocytes and adult dermal fibroblasts under both low- and high-Ca2+ conditions. In all of these assays, responses of sun-protected and sun-exposed skin were identical. In contrast, responses of neonatal foreskin to RA were similar to those of adult skin in the presence of low-Ca2+ culture medium, but under conditions of high extracellular Ca2+ RA provided little or no additional stimulus. Together these studies suggest that the ability of RA to enhance repair of sun-damaged skin (documented in previous studies) may reflect its ability to influence the behavior of skin in a manner that is age dependent but independent of sun-exposure status.

Expression of serine proteinases and metalloproteinases in organ-cultured human skin. Altered levels in the presence of retinoic acid and possible relationship to retinoid-induced loss of epidermal cohesion.

Neonatal human foreskin obtained at circumcision was cut into 2 x 2-mm pieces and placed in organ culture. Culture medium consisted of a serum-free, growth factor-free basal medium containing either 0.15 mmol/L Ca2+ or 1.4 mmol/L Ca2+. Some cultures were left as control, whereas others were treated with 3 mumol/L all-trans retinoic acid (RA). In the presence of RA, epidermal cohesion was disrupted and the upper layers separated from the viable epidermis beneath. This effect was observed under both low Ca2+ and high Ca2+ conditions. At 2-day intervals, culture fluids were collected and analyzed for serine and metalloproteinase activities. Serine proteinase activity was detected in the culture fluids and virtually all of the detected activity was dependent on the presence of plasminogen. Activity was elevated in the RA-treated tissues and this was due to increased amounts of both urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). Elastase and cathepsin G were not detected in either control or RA-treated cultures. Increased plasminogen activator levels were also detected in RA-treated keratinocytes and fibroblasts in monolayer culture. Significant amounts of t-PA (though not u-PA) were found in fibroblast culture fluids, whereas both t-PA and u-PA were detected in culture fluids from keratinocytes. Metalloproteinase activity was also detected in the culture fluids of control and RA-treated tissues but in contrast to plasminogen activator, metalloproteinase activity decreased in the presence of RA. Casein and gelatin zymographic studies indicated the presence of both 92- and 72-kd gelatinases and stromelysin-1 and suggested that the decreased activity was primarily due to reduction in the 92- and 72-kd gelatinases. When serine proteinase inhibitors (aprotinin and soybean trypsin inhibitor) were included in the culture medium throughout the incubation period, epidermal discohesion was reduced. A metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-2, did not have this effect. Taken together, these data show that a number of proteolytic enzymes are produced during organ culture of human skin. They suggest that these proteases may influence the structural integrity of the tissue.

Increased expression of pp60c-src in gastric mucosa of aged rats.

The relationship between proliferative activity and the expression of pp60c-src in gastric mucosa (oxyntic gland area) of young (4-month) and aged (24-month) Fischer 344 rats was examined. Gastric mucosal proliferative activity, as assessed by 5-bromo-2'-deoxyuridine (BrdU) immunoreactive cells, was found to be 115% (p < .001) higher in aged than in young rats. This was associated with a 47% rise (p < .025) in overall tyrosine kinase (Tyr-k) activity and a 5-7-fold increase in autophosphorylation of four prominent protein bands with M(r) of 40, 55, 60, and 70 kDa in gastric mucosal membranes. A similar phenomenon was also observed for Tyr-k activity of pp60c-src in that the aged rats revealed a 69% (p < .025) higher enzyme activity and a 5-fold rise in the extent of autophosphorylation of this protein when compared with the corresponding values from young animals. Increased Tyr-k activity of pp60c-src in the gastric mucosa of aged rats could in part be due to higher levels of this protein because the relative concentration of pp60c-src, as assessed by Western blot analysis, showed a 2-5-fold increase over the young animals. In addition, the relative concentration of c-src mRNA in the gastric mucosa of aged rats was also found to be 5-6-fold higher than in young animals. We suggest that pp60c-src may play a role in regulating gastric mucosal proliferative processes in the gastric mucosa of aged rats.

Aging diminishes gastric mucosal regeneration: relationship to tyrosine kinases.

BACKGROUND: Increased incidence of gastric ulcer observed in the aged could be partly attributed to increased susceptibility of the mucosa to various damaging agents together with impediment of the repair process. The present investigation was undertaken to compare the rate of mucosal regeneration and the role of tyrosine kinases in regulation of this process between young (4-month-old) and aged (24-month-old) rats during the first 24 hours after injury. EXPERIMENTAL DESIGN: Groups of young and aged rats were given intragastrically with either 2 M NaCl (1.5 ml/130 gm body weight), or an equivalent volume of water and killed 1, 6, and 24 hours later. Each animal was injected intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU; 50 mg/kg) 1 hour before killing to assess proliferative activity by immunocytochemistry. The stomach (oxyntic gland area) was also evaluated by light microscopy for the extent of injury and subsequent regeneration, and mucosa assayed for ornithine decarboxylase and tyrosine kinase (Tyr-k) activity and tyrosine phosphorylation of membrane proteins. RESULTS: Although 2 M NaCl caused extensive damage to the gastric mucosa in both young and aged rats, as evidenced by the total loss of the surface epithelium at 1 hour postinjury, the degree of regeneration was faster in young animals. In young rats, gastric epithelium showed signs of regeneration at 6 hours postinjury and was essentially complete by 24 hours. In contrast, in aged rats, only intermittent surface cells were seen 24 hours after injury. In both age groups, injury resulted in stimulation of mucosal proliferative activity. However, whereas ornithine decarboxylase activity in both age groups was maximally stimulated (350% in young versus 80% in aged) at 6 hours after injury, the number of BrdU-positive cells in young rats increased steadily with time after injury. In contrast, aged rats showed a biphasic pattern in that the number of BrdU-positive cells/gland remained decreased for up to 6 hours, whereafter a steep rise occurred. At 24 hours after injury, the number of BrdU-positive cells/gastric gland in aged rats were found to be higher than in young rats (6 +/- 1.5 cells/gland in young rats versus 9 +/- 2.1 cells/gland in aged rats). The pattern of Tyr-k activity in young and aged rats after injury was found to be quite different from that observed for proliferative activity. In young rats, mucosal Tyr-k activity increased by about 60% at 1 hour after injury, then decreased slightly over the next 5 hours and increased again revealing a 120% rise at 24 hours postinjury. This was associated with a concomitant change in tyrosine phosphorylation of six membrane proteins with molecular weight (in kilodalton) of 30, 35, 50, 55, 60 and 70. In contrast, in aged rats, Tyr-k activity was increased only marginally (about 20%) during the first 6 hours, but at 24 hours postinjury it was found to be 70% above the control. In aged rats, injury produced no significant stimulation in tyrosine phosphorylation of gastric mucosal membrane proteins. CONCLUSIONS: We conclude that aging is associated with the diminished regenerative capacity of the gastric mucosa. This could partly be attributed to diminished activation of mucosal Tyr-k and decreased tyrosine phosphorylation of certain membrane proteins.

all-trans-retinoic acid preserves viability of fibroblasts and keratinocytes in full-thickness human skin and fibroblasts in isolated dermis in organ culture.

Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue as the criterion, it was observed that no keratinocytes and few fibroblasts survived incubation for 10-12 days in serum-free basal medium containing a low level (0.15 mM) of extracellular Ca2+. Increasing the extracellular Ca2+ concentration to 1.4 mM or treating the tissue with 3 microM retinoic acid (RA) under low Ca2+ conditions resulted in increased keratinocyte and fibroblast survival; the two treatments together were more effective than either treatment alone. The same treatments preserved fibroblast survival when pieces of isolated dermal tissue were incubated in organ culture and also supported fibroblast survival in monolayer culture. These findings indicate that recovery of keratinocytes and fibroblasts from skin after maintenance in organ culture provides a simple but definitive measure of the viability of the major cellular elements present in the tissue. These findings suggest that RA treatment enhances survival of both fibroblasts and keratinocytes and that these effects of RA can be seen at physiological Ca2+ concentrations as well as at suboptimal levels of extracellular Ca2+. Finally, these results indicate that the dermis is a direct target of RA.