Epidermal growth factor receptor cross-talks with ligand-occupied estrogen receptor-alpha to modulate both lactotroph proliferation and prolactin gene expression.

Both estrogen (E2) and EGF regulate lactotrophs, and we recently demonstrated that EGF phosphorylates S118 on estrogen receptor-alpha (ERalpha) and requires ERalpha to stimulate prolactin (PRL) release. However, the interactions between ligand-occupied ERalpha and activated ErbB1 and its impact on lactotroph function are unknown. Using rat GH3 lactotrophs, we found that both E2 and EGF independently stimulated proliferation and PRL gene expression. Furthermore, their combination resulted in an enhanced stimulatory effect on both cell proliferation and PRL gene expression. Inhibitors of ER as well as ErbB1 blocked the combined effects of E2 and EGF. Pretreatment with UO126 abolished the combined effects, demonstrating Erk1/2 requirement. Although bidirectionality in ER-ErbB1 cross-talk is a well-accepted paradigm, interestingly in lactotrophs, ErbB1 kinase inhibitor failed to block the effect of E2 on proliferation and stimulation of PRL gene expression, suggesting that ER does not require ErbB1 to mediate its effects. Furthermore, E2 did not affect the ability of EGF to induce c-Fos expression or modulate AP-1 activity. However, both E2 and EGF combine to enhance S118 phosphorylation of ERalpha, leading to enhanced E2-mediated estrogen response element transactivation. Taken together, our results suggest that, in lactotrophs, activated ErbB1 phosphorylates ERalpha to enhance the stimulatory effect of E2, thereby providing the molecular basis by which EGF amplifies the response of E2.

Curcumin (diferuloylmethane) inhibits cell proliferation, induces apoptosis, and decreases hormone levels and secretion in pituitary tumor cells.

Prolactinomas are the most prevalent functional pituitary adenomas. Dopamine D2 receptor (D2R) agonists, such as bromocriptine are the first line of therapy; however, drug intolerance/resistance to D2R agonists exists. Apart from D2R agonists, there is no established medical therapy for prolactinomas; therefore, identifying novel therapeutics is warranted. Curcumin, a commonly used food additive in South Asian cooking, inhibits proliferation of several tumor cell lines; however, its effect on pituitary tumor cell proliferation has not been determined. Our objectives were to: 1) determine whether curcumin inhibits proliferation of pituitary tumor cell lines; 2) identify the signaling intermediaries that mediate the effect of curcumin; 3) examine whether curcumin inhibited pituitary hormone production and release; and 4) examine whether curcumin could enhance the growth-inhibitory effect of bromocriptine. Using rat lactotroph cell lines, GH3 and MMQ cells, we report that curcumin had a robust dose and time-dependent inhibitory effect on GH3 and MMQ cell proliferation. Inhibitory effects of curcumin persisted, even on removal of curcumin, and curcumin also blocked colony formation ability of pituitary tumor cells. The growth-inhibitory effect of curcumin was accompanied by decreased expression of cyclin D3 and ser 780 phosphorylation of retinoblastoma protein. In addition, curcumin also induced apoptosis in both GH3 and MMQ cells. Furthermore, curcumin suppresses intracellular levels and release of both prolactin and GH. Finally, we show that low concentrations of curcumin enhanced the growth-inhibitory effect of bromocriptine on MMQ cell proliferation. Taken together we demonstrate that curcumin inhibits pituitary tumor cell proliferation, induces apoptosis, and decreases hormone production and release, and thus, we propose developing curcumin as a novel therapeutic tool in the management of prolactinomas.

Platelets from diabetic pigs exhibit hypersensitivity to thrombin.

Responses of platelets from diabetic and diabetic-hyperlipidemic pigs were studied. Pigs were made diabetic with single dose of alloxan, which acts by selectively destroying insulin-producing pancreatic beta cells thus inducing type 1 diabetes. Pigs were kept for 1 or 12 wk, during which thrombin-induced aggregation was monitored in washed platelets. The platelets showed increased sensitivity to aggregation within 1 wk of induction of diabetes. Hyperlipidemia alone for 12 wk did not increase platelet hypersensitivity, but hyperlipidemia together with diabetes significantly increased thrombin-induced platelet aggregation. Because this hypersensitivity occurred in washed platelets, this characteristic appears to be independent of any contribution by plasma factors or other blood cells. The hypersensitivity of platelets from diabetic pigs correlated with decreased activity of mitogen-activated protein kinase. These studies offer the first evidence that platelet hyperactivity occurs during the early stages (within a week) of induction of diabetes in pigs and before manifestation of other cardiovascular problems.

Autocrine extracellular signal-regulated kinase (ERK) activation in normal human keratinocytes: metalloproteinase-mediated release of amphiregulin triggers signaling from ErbB1 to ERK.

ErbB signaling through extracellular signal-regulated kinase (ERK) has been implicated in regulating the expression of ErbB ligands in hyperproliferative skin disorders and wound healing. Here, we characterize the process of autocrine ERK activation in cultured normal human keratinocytes (NHKs) subjected to growth factor (GF) deprivation. Basal ERK phosphorylation was lower after 48 h than after 24 h of GF deprivation, and lowest at 30-60 min after an additional medium change. ERK phosphorylation was markedly increased by low concentrations of epidermal growth factor (EGF) (0.2-1 ng/ml) that provoked only a limited increase in ErbB1 tyrosine phosphorylation and internalization. Basal ErbB tyrosine phosphorylation and ERK phosphorylation were inhibited by two different ErbB receptor tyrosine kinase inhibitors, by the ErbB1-specific neutralizing monoclonal antibody 225 IgG, by two different metalloproteinase inhibitors, and by neutralizing antibodies against amphiregulin (AR). In contrast, these responses were unaffected by neutralizing antibodies against other ErbB1 ligands or the ErbB2 inhibitors geldanamycin and AG825. The time course of autocrine ERK phosphorylation correlated with the appearance of soluble AR, and two different metalloproteinase inhibitors blocked AR release. These results define an amphiregulin- and ErbB1-dependent mechanism by which autocrine ERK activation is maintained in NHKs, even when ErbB1 autophosphorylation and internalization are limited.

Comparative potency of obeticholic acid and natural bile acids on FXR in hepatic and intestinal in vitro cell models.

Obeticholic acid (OCA) is a semisynthetic farnesoid X receptor (FXR) agonist, an analogue of chenodeoxycholic acid (CDCA) which is indicated for the treatment of primary biliary cholangitis (PBC) in combination with ursodeoxycholic acid (UDCA). OCA efficiently inhibits bile acid synthesis and promotes bile acid efflux via activating FXR-mediated mechanisms in a physiologically relevant in vitro cell system, Sandwich-cultured Transporter Certified ™ human primary hepatocytes (SCHH). The study herein evaluated the effects of UDCA alone or in combination with OCA in SCHH. UDCA (≤100 µmol/L) alone did not inhibit CYP7A1 mRNA, and thus, no reduction in the endogenous bile acid pool observed. UDCA ≤100 µmol/L concomitantly administered with 0.1 µmol/L OCA had no effect on bile acid synthesis beyond what was observed with OCA alone. Furthermore, this study evaluated human Caco-2 cells (clone C2BBe1) as in vitro intestinal models. Glycine conjugate of OCA increased mRNA levels of FXR target genes in Caco-2 cells, FGF-19, SHP, OSTα/ß, and IBABP, but not ASBT, in a concentration-dependent manner, while glycine conjugate of UDCA had no effect on the expression of these genes. The results suggested that UDCA ≤100 µmol/L did not activate FXR in human primary hepatocytes or intestinal cell line Caco-2. Thus, co-administration of UDCA with OCA did not affect OCA-dependent pharmacological effects.

Differential effects of estrogen receptor antagonists on pituitary lactotroph proliferation and prolactin release.

Anti-estrogens act by inhibiting estrogen receptor (ER) function. Unlike raloxifene and tamoxifen which exhibit both antagonist and agonist properties, ICI 182,780 (ICI) is considered a "pure" anti-estrogen devoid of any agonistic activities. Whereas there is ample information on the effects of anti-estrogens on the breast and uterus, little is known about their action on the pituitary, the estrogen-sensitive master endocrine gland. Our objectives were to: (1) compare the effects of ICI, tamoxifen and raloxifene on lactotroph proliferation in the absence of estrogen, (2) determine whether their action is mediated through the ER, and (3) compare their effects on prolactin (PRL) release. We are reporting that ICI is a potent inhibitor of lactotroph proliferation (both GH3 and MMQ cells) with maximal inhibition of 45-50% seen with 1nM. ICI is several orders of magnitude more potent than raloxifene while tamoxifen has no effect. Neither anti-estrogen affects T47D breast cancer cell proliferation. GH3 cell incubation with ICI for 1h only causes maximal suppression of cell proliferation, an effect which is reversed by co-incubation with estrogen. Such a short exposure to ICI is sufficient to cause rapid and persistent downregulation of ERalpha protein, whereas downregulation of ERbeta is significantly delayed; tamoxifen and raloxifene have no appreciable effects on ER(s) levels. The ability of ICI to inhibit GH3 cell proliferation is dependent upon ERalpha, since an ERalpha, but not ERbeta, specific agonist reverses the effect of ICI. PRL release is differentially regulated by the anti-estrogens. ICI at 0.1nM suppresses PRL release from GH3 cells by 80%, with a similar strong suppression also seen with 10nM raloxifene. However, tamoxifen at 0.01nM inhibits PRL release but has no effect at 10nM. Cell co-incubation with ICI and estradiol results in a four-fold increase in PRL release. Taken together, our study shows that ICI, in the absence of exogenous estrogens, inhibits lactotroph proliferation and PRL release by downregulating or inactivating ERalpha. The dissimilar responses of cell proliferation and PRL release to the anti-estrogens suggest that both processes are regulated by different mechanisms. These data highlight the importance of studying the effects of anti-estrogens in multiple systems.

Biobreeding rat islets exhibit reduced antioxidative defense and N-acetyl cysteine treatment delays type 1 diabetes.

Islet-level oxidative stress has been proposed as a trigger for type 1 diabetes (T1D), and release of cytokines by infiltrating immune cells further elevates reactive oxygen species (ROS), exacerbating ß cell duress. To identify genes/mechanisms involved with diabetogenesis at the ß cell level, gene expression profiling and targeted follow-up studies were used to investigate islet activity in the biobreeding (BB) rat. Forty-day-old spontaneously diabetic lymphopenic BB DRlyp/lyp rats (before T cell insulitis) as well as nondiabetic BB DR+/+ rats, nondiabetic but lymphopenic F344lyp/lyp rats, and healthy Fischer (F344) rats were examined. Gene expression profiles of BB rat islets were highly distinct from F344 islets and under-expressed numerous genes involved in ROS metabolism, including glutathione S-transferase (GST) family members (Gstm2, Gstm4, Gstm7, Gstt1, Gstp1, and Gstk1), superoxide dismutases (Sod2 and Sod3), peroxidases, and peroxiredoxins. This pattern of under-expression was not observed in brain, liver, or muscle. Compared with F344 rats, BB rat pancreata exhibited lower GST protein levels, while plasma GST activity was found significantly lower in BB rats. Systemic administration of the antioxidant N-acetyl cysteine to DRlyp/lyp rats altered abundances of peripheral eosinophils, reduced severity of insulitis, and significantly delayed but did not prevent diabetes onset. We find evidence of ß cell dysfunction in BB rats independent of T1D progression, which includes lower expression of genes related to antioxidative defense mechanisms during the pre-onset period that may contribute to overall T1D susceptibility.

Growth suppression of mouse pituitary corticotroph tumor AtT20 cells by curcumin: a model for treating Cushing's disease.

BACKGROUND: Pituitary corticotroph tumors secrete excess adrenocorticotrophic hormone (ACTH) resulting in Cushing's disease (CD). Standard treatment includes surgery and, if not successful, radiotherapy, both of which have undesirable side effects and frequent recurrence of the tumor. Pharmacotherapy using PPARgamma agonists, dopamine receptor agonists, retinoic acid or somatostatin analogs is still experimental. Curcumin, a commonly used food additive in South Asian cooking, has potent growth inhibitory effects on cell proliferation. Our laboratory recently demonstrated that curcumin inhibited growth and induced apoptosis in prolactin- and growth hormone-producing tumor cells. Subsequently, Schaaf et.al. confirmed our findings and also showed the in vivo effectiveness of curcumin to suppress pituitary tumorigenesis. However the molecular mechanism that mediate this effect of curcumin are still unknown. PRINCIPAL FINDINGS: Using the mouse corticotroph tumor cells, AtT20 cells, we report that curcumin had a robust, irreversible inhibitory effect on cell proliferation and clonogenic property. The curcumin-induced growth inhibition was accompanied by decreased NFkappaB activity. Further, curcumin down-regulated the pro-survival protein Bcl-xL, depolarized the mitochondrial membrane, increased PARP cleavage, which led to apoptotic cell death. Finally, curcumin had a concentration-dependent suppressive effect on ACTH secretion from AtT20 cells. CONCLUSION: The ability of curcumin to inhibit NFkappaB and induce apoptosis in pituitary corticotroph tumor cells leads us to propose developing it as a novel therapeutic agent for the treatment of CD.

Curcumin (diferuloylmethane) induces apoptosis and blocks migration of human medulloblastoma cells.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Bcl-2 and MMP-9 promote the pathogenesis and progression of MB. The expression of both bcl-2 and MMP-9 is regulated by the transcription factor NF-kappaB. Curcumin, a natural food additive, has a potent anti-proliferative effect, presumably mediated through NF-kappaB suppression. The tumor-suppressing effects of curcumin are well documented, however, its effect on MB is unknown. Our objectives were to: a) examine the effect of curcumin on MB cell proliferation and apoptosis; b) characterize the mechanism that mediates the effect of curcumin; c) examine the effects of curcumin on MB cell migration. We report that curcumin inhibited cell proliferation and blocked clonogenicity of MB cells. Furthermore, curcumin down-regulated bcl-2 and bcl(x)l, leading to caspase-mediated cell death. Finally, curcumin blocked migration of MB cells. Thus, we propose developing curcumin as a novel therapeutic agent for MB.

Selective estrogen receptor down-regulator and selective estrogen receptor modulators differentially regulate lactotroph proliferation.

BACKGROUND: We recently reported that estrogen receptor alpha (ERalpha), even in absence of estrogen (E2), plays a critical role in lactotroph homeostasis. The anti-estrogen ICI 182780 (ICI), but not tamoxifen or raloxifene, rapidly promoted the degradation of ERalpha, and inhibited cell proliferation. However, all three ER antagonists suppressed PRL release, suggesting that receptor occupation is sufficient to inhibit prl gene expression whereas receptor degradation is required to suppress lactotroph proliferation. In this study our objective was to determine whether ERalpha degradation versus occupation, differentially modulates the biological outcome of anti-estrogens. PRINCIPAL FINDINGS: Using the rat lactotroph cell line, GH3 cells, we report that ICI induced proteosome mediated degradation of ERalpha. In contrast, an ERalpha specific antagonist, MPP, that does not promote degradation of ERalpha, did not inhibit cell proliferation. Further, ICI, but not MPP, abolished anchorage independent growth of GH3 cells. Yet, both ICI and MPP were equally effective in suppressing prl expression and release, as well as ERE-mediated transcriptional activity. CONCLUSION: Taken together, our results demonstrate that in lactotrophs, ERalpha degradation results in decreased cell proliferation, whereas ERalpha occupation by an antagonist that does not promote degradation of ERalpha is sufficient to inhibit prl expression.

Identification of a serum-induced transcriptional signature associated with type 1 diabetes in the BioBreeding rat.

OBJECTIVE: Inflammatory mediators associated with type 1 diabetes are dilute and difficult to measure in the periphery, necessitating development of more sensitive and informative biomarkers for studying diabetogenic mechanisms, assessing preonset risk, and monitoring therapeutic interventions. RESEARCH DESIGN AND METHODS: We previously utilized a novel bioassay in which human type 1 diabetes sera were used to induce a disease-specific transcriptional signature in unrelated, healthy peripheral blood mononuclear cells (PBMCs). Here, we apply this strategy to investigate the inflammatory state associated with type 1 diabetes in biobreeding (BB) rats. RESULTS: Consistent with their common susceptibility, sera of both spontaneously diabetic BB DRlyp/lyp and diabetes inducible BB DR+/+ rats induced transcription of cytokines, immune receptors, and signaling molecules in PBMCs of healthy donor rats compared with control sera. Like the human type 1 diabetes signature, the DRlyp/lyp signature, which is associated with progression to diabetes, was differentiated from that of the DR+/+ by induction of many interleukin (IL)-1-regulated genes. Supplementing cultures with an IL-1 receptor antagonist (IL-1Ra) modulated the DRlyp/lyp signature (P < 10(-6)), while administration of IL-1Ra to DRlyp/lyp rats delayed onset (P = 0.007), and sera of treated animals did not induce the characteristic signature. Consistent with the presence of immunoregulatory cells in DR+/+ rats was induction of a signature possessing negative regulators of transcription and inflammation. CONCLUSIONS: Paralleling our human studies, serum signatures in BB rats reflect processes associated with progression to type 1 diabetes. Furthermore, these studies support the potential utility of this approach to detect changes in the inflammatory state during therapeutic intervention.

Estrogen receptor-alpha mediates the epidermal growth factor-stimulated prolactin expression and release in lactotrophs.

Epidermal growth factor (EGF) is a potent regulator of cell function in many cell types. EGF-receptor (EGFR/ErbB1)-activated Erk1/2 has been reported to activate estrogen receptor (ER) in an estrogen (E2)-independent manner. In the pituitary lactotrophs, both EGF and E2 stimulate prolactin (PRL) release, but the nature of interactions between ErbB and ERalpha signaling is unknown. Our objectives were to 1) characterize EGF-induced PRL release, 2) determine whether this effect requires ERalpha, and 3) determine the molecular basis for cross talk between ErbB and ERalpha signaling pathways. Using GH3 cells, a rat lactotroph cell line, we report that EGF stimulates PRL gene expression and release in a dose- and time-dependent manner. EGF caused a rapid and robust activation of Erk1/2 via ErbB1 and induced phosphorylation of S118 on ERalpha in an Erk1/2-dependent manner. The global antiestrogen ICI 182780 and the ERalpha-specific antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylet hoxy)phenol]-1H-pyrazole dihydrochloride (MPP), but not the ERbeta-specific antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), blocked the EGF-induced PRL release, indicating an ERalpha requirement. This was further supported by using ERalpha knockdown by small interfering RNA. Because the antiestrogens did not block EGF-induced Mek-1 or Erk1/2 phosphorylation, ERalpha is placed downstream from the ErbB1-activated Erk1/2. These results provide the first evidence that ErbB1-induced PRL release is ERalpha dependent.

Differential ErbB1 signaling in squamous cell versus basal cell carcinoma of the skin.

In this study, we examined ErbB1 signaling in human basal and squamous cell carcinomas (BCC and SCC) of the skin in vivo. We used enzyme-linked immunosorbent assay, laser capture microdissection-coupled real-time reverse transcriptase-polymerase chain reaction, and immunohistochemistry to assess expression and activation levels of ErbB1 protein, ligands, and potential downstream effectors, in BCC and SCC tumors, stroma, and adjacent epidermis. Although total ErbB1 protein and mRNA were similar in cancerous and normal skin, we found that ErbB1 activation (phospho-Tyr(1068)) was greater in bulk SCC versus BCC or normal skin. In addition, three ErbB1 ligand transcripts (amphiregulin, heparin-binding epidermal growth factor-like growth factor, and transforming growth factor-alpha) were up-regulated in tumor cells of SCC but not BCC. Expression of these ligands was also increased in asymptomatic epidermis adjacent to both SCC and BCC, relative to normal skin. Interestingly, betacellulin transcript levels were inversely regulated compared with the other ligands. Consistently, downstream ErbB1 effectors (Erk1/2 and Akt) were activated in tumor cells of SCC but not of BCC and in adjacent epidermis of both BCC and SCC. These results demonstrate that ErbB1 signaling is hyperactive in tumor cells of SCC but not of BCC and in nearby asymptomatic epidermis of both tumor types. Our results suggest that targeting ErbB1 signaling might be of benefit in the treatment of SCC.

Src family kinase inhibitors block amphiregulin-mediated autocrine ErbB signaling in normal human keratinocytes.

c-Src potentiates proliferation, survival, and invasiveness in response to epidermal growth factor (EGF) in human mammary carcinoma cells. Tyrosine (Tyr) 845 of ErbB1 is phosphorylated by Src and has been implicated in control of malignant behavior. Although several lines of evidence also suggest important interactions of ErbB and Src family kinase signaling in normal epithelial cells, little is known about the mechanism of this interaction. Studying normal human keratinocytes (NHKs), here we demonstrate strong expression of the Src family kinases Src, Yes, and Fyn; Src family kinase-dependent stimulation of Tyr 845 by EGF; and potent inhibition of NHK proliferation and migration by two Src family kinase inhibitors PP1 and PD173952. EGF-stimulated extracellular signal-regulated kinase (ERK) phosphorylation occurred at much lower concentrations of EGF than required to phosphorylate Tyr 845. Moreover, the effect of Src family kinase inhibitors on EGF-stimulated ERK phosphorylation was transient, prompting a search for other targets of Src family kinase action. By enzyme-linked immunosorbent assay analysis, we found that three different Src family kinase inhibitors [6-(2,6-dichlorophenyl)-8-methyl-2-(4-morpholin-4-ylphenylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (PD173952), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), and 2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (SU6656)] markedly inhibited elaboration of soluble amphiregulin by NHKs. The ErbB inhibitor PD158780 and the mitogen-activated protein kinase kinase inhibitor U0126 also markedly inhibited NHK proliferation, migration, and amphiregulin production. Together, these observations demonstrate that one or more Src family kinases act upstream as well as downstream of ErbB1 to promote amphiregulin-dependent autocrine stimulation of NHKs and suggest that autocrine NHK proliferation is more dependent upon ERK activation than upon Tyr 845 phosphorylation.

Metalloproteinases stimulate ErbB-dependent ERK signaling in human skin organ culture.

To investigate the role of ERK signaling in human skin responses to wounding, organ cultures of human skin were maintained for 0.5-24 h in the presence of various inhibitors, followed by measurement of ERK phosphorylation or mRNA levels. The MEK inhibitor PD98059 produced near-complete (97-98%) inhibition of ERK phosphorylation, whereas inhibition of c-Fos, c-Jun, HB-EGF, AR, and VEGF mRNA by this compound was incomplete (41-65%). PD98059 was significantly more effective than either PD158780 or BB2516 as an inhibitor of ERK phosphorylation and of the rapid rise in c-Fos and c-Jun mRNA expression. In contrast, all three compounds inhibited the more delayed rise in HB-EGF mRNA to the same extent. Exogenous epidermal growth factor abrogated the inhibition of ERK phosphorylation caused by BB2516. These data indicate that one or more metalloproteinases activate ErbB signaling in skin organ culture, that ErbB signaling plays an important but not exclusive role in the activation of ERK, and that non-ERK pathways contribute to gene expression in this system. Because metalloproteinase-mediated cleavage of the HB-EGF transmembrane precursor is known to be ERK-dependent, our data suggest that ERK activation resulting from initial trauma leads to metalloproteinase-mediated cleavage of HB-EGF, thereby triggering the ErbB signaling cascade.

Keratinocyte outgrowth from human skin explant cultures is dependent upon p38 signaling.

Organ culture of skin is known to recapitulate several early events in the process of wound healing. Here we investigate the function of p38 kinase signaling as a regulator of keratinocyte behavior in human skin organ culture. We first show that skin organ culture recapitulates the transition from migration to proliferation that is known to characterize the reepithelialization process. We next show that inhibition of p38 markedly impairs the formation of keratinocyte outgrowth in human skin explant cultures, as well as the migration of keratinocytes in an in vitro wound assay. In contrast, the marked induction of mRNA encoding the ErbB ligand heparin-binding epidermal growth factor-like growth factor, known to occur after skin wounding, was not blocked by inhibition of p38. As assessed by immunoblotting, phosphorylation of p38 was limited and was not increased between 0 and 7 days of organ culture. Our results show the sensitivity of reepithelialization to inhibition by p38 and suggest that p38 acts primarily during the migration phase of this process. These data also indicate that autocrine heparin-binding epidermal growth factor expression is not regulated by p38.

Differential cytoskeletal association of ErbB1 and ErbB2 during keratinocyte differentiation.

ErbB1 and ErbB2 display differential subcellular localization in human skin and cultured keratinocytes. To determine whether ErbB1 and ErbB2 also differ in cytoskeletal binding properties, normal human keratinocytes grown under conditions favoring a basal or differentiated phenotype were repeatedly extracted in a non-ionic detergent buffer. In basaloid keratinocytes, cytoskeletal association of ErbB1 and ErbB2 was limited. ErbB1 ( approximately 5%) was tightly associated with the cytoskeleton, compared to <1% of ErbB2 (p=0.004). After EGF stimulation, activated ErbB1 and ERK associated with the cytoskeleton to a greater extent than did total ErbB1 and total ERK. Association of ErbB2 increased markedly in differentiated keratinocytes, whereas association of ErbB1 was similar in basaloid and differentiated cells. Cytoskeletal association of ErbB2 correlated with plasma membrane localization. These results suggest that ErbB1 and ErbB2 employ different mechanisms of plasma membrane targeting during keratinocyte differentiation, and that cytoskeletal association may facilitate the coupling of activated ErbB1 and ERK.

Insulin-like growth factor-I has a biphasic effect on colon carcinoma cells through transient inactivation of forkhead1, initially mitogenic, then mediating growth arrest and differentiation.

IGF-I stimulates intestinal cell differentiation after initiating a short proliferative burst, similar to its effect on muscle cell differentiation. Levels of IGF-I attainable in serum (10-20 ng/ml) induced transient growth stimulation of colon carcinoma cells, then growth arrest. When IGF-I functioned as a mitogen, it blocked differentiation. Intestinal cell differentiation occurred once cells had undergone the IGF-I-initiated growth arrest and IGF-I and butyrate acted synergistically to induce maturation markers. IGF-I induces NIH-3T3 cell proliferation and survival by activating the kinase akt, which in turn inhibits various apoptotic mediators and the forkhead family of transcription factors, which mediate expression of p27(kip1). Promoter reporter assays demonstrated that forkhead1 mediates transcription of p27(kip1) in colon carcinoma cells. The mitogenic effects of IGF-I on 4 colon carcinoma cell lines were transient because the inactivating phosphorylation of forkhead1 by akt was short-lived. This allowed transcriptional upregulation of the cdk inhibitor p27(kip1), with a resulting growth arrest. In contrast, in NIH-3T3 cells treated in parallel with identical IGF-I levels, forkhead phosphorylation levels were sustained; thus, no increase in p27(kip1) levels was seen and cells continued to proliferate. Intestinal epithelial cells in vivo undergo a limited number of divisions, then growth arrest and completion of their maturation. IGFs found in intestinal tissue may control the timing of this process. In addition, colon cancers may have developed strategies to overcome IGF-I-mediated growth arrest. Earlier (Kansra et al., Int J Cancer 2000;87:373-8), we found that levels of IGFBP-3 were elevated at least 2-fold in 70% of resected colon cancers compared with adjacent normal tissue. In the current study, growth inhibition by IGF-I and IGF-II was blocked by concurrent addition of IGFBP-3, implying that colon cancers with elevated IGFBP-3 levels would be selected for in vivo because they could bind and inactivate high serum IGF-I levels and continue to proliferate.