IL-6 receptor blockade does not slow ß cell loss in new-onset type 1 diabetes.

BackgroundIL-6 receptor (IL-6R) signaling drives development of T cell populations important to type 1 diabetes pathogenesis. We evaluated whether blockade of IL-6R with monoclonal antibody tocilizumab would slow loss of residual ß cell function in newly diagnosed type 1 diabetes patients.MethodsWe conducted a multicenter, randomized, placebo-controlled, double-blind trial with tocilizumab in new-onset type 1 diabetes. Participants were screened within 100 days of diagnosis. Eligible participants were randomized 2:1 to receive 7 monthly doses of tocilizumab or placebo. The primary outcome was the change from screening in the mean AUC of C-peptide collected during the first 2 hours of a mixed meal tolerance test at week 52 in pediatric participants (ages 6-17 years).ResultsThere was no statistical difference in the primary outcome between tocilizumab and placebo. Immunophenotyping showed reductions in downstream signaling of the IL-6R in T cells but no changes in CD4 memory subsets, Th17 cells, Tregs, or CD4+ T effector cell resistance to Treg suppression. A DC subset decreased during therapy but regressed to baseline once therapy stopped. Tocilizumab was well tolerated.ConclusionTocilizumab reduced T cell IL-6R signaling but did not modulate CD4+ T cell phenotypes or slow loss of residual ß cell function in newly diagnosed individuals with type 1 diabetes.Trial RegistrationClinicalTrials.gov NCT02293837.FundingNIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and National Institute of Allergy and Infectious Diseases (NIAID) UM1AI109565, UL1TR000004 from NIH/National Center for Research Resources (NCRR) Clinical and Translational Science Award (CTSA), NIH/NIDDK P30DK036836, NIH/NIDDK U01DK103266, NIH/NIDDK U01DK103266, 1UL1TR000064 from NIH/NCRR CTSA, NIH/National Center for Advancing Translational Sciences (NCATS) UL1TR001878, UL1TR002537 from NIH/CTSA; National Health and Medical Research Council Practitioner Fellowship (APP1136735), NIH/NIDDK U01-DK085476, NIH/CTSA UL1-TR002494, Indiana Clinical and Translational Science Institute Award UL1TR002529, Vanderbilt Institute for Clinical and Translational Research UL1TR000445. NIH/NCATS UL1TR003142, NIH/CTSA program UL1-TR002494, Veteran Affairs Administration, and 1R01AI132774.

The risk of progression to type 1 diabetes is highly variable in individuals with multiple autoantibodies following screening.

AIMS/HYPOTHESIS: Young children who develop multiple autoantibodies (mAbs) are at very high risk for type 1 diabetes. We assessed whether a population with mAbs detected by screening is also at very high risk, and how risk varies according to age, type of autoantibodies and metabolic status. METHODS: Type 1 Diabetes TrialNet Pathway to Prevention participants with mAbs (n = 1815; age, 12.35 ± 9.39 years; range, 1-49 years) were analysed. Type 1 diabetes risk was assessed according to age, autoantibody type/number (insulin autoantibodies [IAA], glutamic acid decarboxylase autoantibodies [GADA], insulinoma-associated antigen-2 autoantibodies [IA-2A] or zinc transporter 8 autoantibodies [ZnT8A]) and Index60 (composite measure of fasting C-peptide, 60 min glucose and 60 min C-peptide). Cox regression and cumulative incidence curves were utilised in this cohort study. RESULTS: Age was inversely related to type 1 diabetes risk in those with mAbs (HR 0.97 [95% CI 0.96, 0.99]). Among participants with 2 autoantibodies, those with GADA had less risk (HR 0.35 [95% CI 0.22, 0.57]) and those with IA-2A had higher risk (HR 2.82 [95% CI 1.76, 4.51]) of type 1 diabetes. Those with IAA and GADA had only a 17% 5 year risk of type 1 diabetes. The risk was significantly lower for those with Index60 <1.0 (HR 0.23 [95% CI 0.19, 0.30]) vs those with Index60 values ≥1.0. Among the 12% (225/1815) ≥12.0 years of age with GADA positivity, IA-2A negativity and Index60 <1.0, the 5 year risk of type 1 diabetes was 8%. CONCLUSIONS/INTERPRETATION: Type 1 diabetes risk varies substantially according to age, autoantibody type and metabolic status in individuals screened for mAbs. An appreciable proportion of older children and adults with mAbs appear to have a low risk of progressing to type 1 diabetes at 5 years. With this knowledge, clinical trials of type 1 diabetes prevention can better target those most likely to progress.

Hepatocyte ERBB3 and EGFR are required for maximal CCl4-induced liver fibrosis.

Epidermal growth factor receptor (EGFR) and its ligands have been implicated in liver fibrosis. However, it has not been directly shown that hepatocellular genetic ablation of either this receptor tyrosine kinase or ERBB3, its interactive signaling partner, affects hepatic fibrosis. Carbon tetrachloride (CCl4)-induced liver fibrosis in hepatocyte-specific (HS) mouse models of EGFR and ERBB3 ablation was evaluated in both single gene knockouts and an HS-EGFR-ERBB3 double knockout (DKO). Loss of hepatocellular EGFR or ERBB3 did not impact cytochrome P450-2E1 expression, the extent of centrilobular injury, or the initial regenerative response, but it did diminish liver fibrosis induced by chronic intraperitoneal administration of CCl4 The reduction of liver fibrosis correlated with reduced α-smooth muscle actin expression. Maximal impact to fibrogenesis occurred in the ERBB3 and EGFR-ERBB3 DKO models, suggesting that EGFR-ERBB3 heterodimeric signaling in damaged hepatocytes may play a more important role in liver fibrosis than EGFR-EGFR homodimeric signaling. Immunohistochemical analyses of phospho-EGFR and phospho-ERBB3 isoforms revealed clear staining in hepatocytes, activated stellate cells, and macrophages. Our results support a role for the hepatocellular ERBB tyrosine kinases in fibrogenesis and suggest that pharmacologic inhibition of EGFR-ERBB3 signaling may reverse or retard hepatic fibrosis.

Loss of hepatocyte ERBB3 but not EGFR impairs hepatocarcinogenesis.

Epidermal growth factor receptor (EGFR) and ERBB3 have been implicated in hepatocellular carcinogenesis (HCC). However, it is not known whether altering the activity of either EGFR or ERBB3 affects HCC development. We now show that Egfr(Dsk5) mutant mice, which have a gain-of-function allele that increases basal EGFR kinase activity, develop spontaneous HCC by 10 mo of age. Their tumors show increased activation of EGFR, ERBB2, and ERBB3 as well as AKT and ERK1,2. Hepatocyte-specific models of EGFR and ERBB3 gene ablation were generated to evaluate how the loss of these genes affected tumor progression. Loss of either receptor tyrosine kinase did not alter liver development or regenerative liver growth following carbon tetrachloride injection. However, using a well-characterized model of HCC in which N-nitrosodiethylamine is injected into 14-day-old mice, we discovered that loss of hepatocellular ERBB3 but not EGFR, which occurred after tumor initiation, retarded liver tumor formation and cell proliferation. We found no evidence that this was due to increased apoptosis or diminished phosphatidylinositol-3-kinase activity in the ERBB3-null cells. However, the relative amount of phospho-STAT3 was diminished in tumors derived from these mice, suggesting that ERBB3 may promote HCC through STAT3 activation.

Loss of hepatocyte EGFR has no effect alone but exacerbates carbon tetrachloride-induced liver injury and impairs regeneration in hepatocyte Met-deficient mice.

The role(s) of the epidermal growth factor receptor (EGFR) in hepatocytes is unknown. We generated a murine hepatocyte specific-EGFR knockout (KO) model to evaluate how loss of hepatocellular EGFR expression affects processes such as EGF clearance, circulating EGF concentrations, and liver regeneration following 70% resection or CCl4-induced centrilobular injury. We were able to disrupt EGFR expression effectively in hepatocytes and showed that the ability of EGF and heregulin (HRG) to phosphorylate EGFR and ERBB3, respectively, required EGFR. Loss of hepatocellular EGFR impaired clearance of exogenous EGF from the portal circulation but paradoxically resulted in reduced circulating levels of endogenous EGF. This was associated with decreased submandibular salivary gland production of EGF. EGFR disruption did not result in increased expression of other ERBB proteins or Met, except in neonatal mice. Liver regeneration following 70% hepatectomy revealed a mild phenotype, with no change in cyclin D1 expression and slight differences in cyclin A expression compared with controls. Peak 5-bromo-2'-deoxyuridine labeling was shifted from 36 to 48 h. Centrilobular damage and regenerative response induced by carbon tetrachloride (CCl4) were identical in the KO and wild-type mice. In contrast, loss of Met increased CCl4-induced necrosis and delayed regeneration. Although loss of hepatocellular EGFR alone did not have an effect in this model, EGFR-Met double KOs displayed enhanced necrosis and delayed liver regeneration compared with Met KOs alone. This suggests that EGFR and Met may partially compensate for the loss of the other, although other compensatory mechanisms can be envisioned.

Epidermal growth factor receptor plays a role in the regulation of liver and plasma lipid levels in adult male mice.

Dsk5 mice have a gain of function in the epidermal growth factor receptor (EGFR), caused by a point mutation in the kinase domain. We analyzed the effect of this mutation on liver size, histology, and composition. We found that the livers of 12-wk-old male Dsk5 heterozygotes (+/Dsk5) were 62% heavier compared with those of wild-type controls (+/+). The livers of the +/Dsk5 mice compared with +/+ mice had larger hepatocytes with prominent, polyploid nuclei and showed modestly increased cell proliferation indices in both hepatocytes and nonparenchymal cells. An analysis of total protein, DNA, and RNA (expressed relative to liver weight) revealed no differences between the mutant and wild-type mice. However, the livers of the +/Dsk5 mice had more cholesterol but less phospholipid and fatty acid. Circulating cholesterol levels were twice as high in adult male +/Dsk5 mice but not in postweaned young male or female mice. The elevated total plasma cholesterol resulted mainly from an increase in low-density lipoprotein (LDL). The +/Dsk5 adult mouse liver expressed markedly reduced protein levels of LDL receptor, no change in proprotein convertase subtilisin/kexin type 9, and a markedly increased fatty acid synthase and 3-hydroxy-3-methyl-glutaryl-CoA reductase. Increased expression of transcription factors associated with enhanced cholesterol synthesis was also observed. Together, these findings suggest that the EGFR may play a regulatory role in hepatocyte proliferation and lipid metabolism in adult male mice, explaining why elevated levels of EGF or EGF-like peptides have been positively correlated to increased cholesterol levels in human studies.

Mitochondrial reactive oxygen species mediate GPCR-induced TACE/ADAM17-dependent transforming growth factor-alpha shedding.

Epidermal growth factor receptor (EGFR) activation by GPCRs regulates many important biological processes. ADAM metalloprotease activity has been implicated as a key step in transactivation, yet the regulatory mechanisms are not fully understood. Here, we investigate the regulation of transforming growth factor-alpha (TGF-alpha) shedding by reactive oxygen species (ROS) through the ATP-dependent activation of the P2Y family of GPCRs. We report that ATP stimulates TGF-alpha proteolysis with concomitant EGFR activation and that this process requires TACE/ADAM17 activity in both murine fibroblasts and CHO cells. ATP-induced TGF-alpha shedding required calcium and was independent of Src family kinases and PKC and MAPK signaling. Moreover, ATP-induced TGF-alpha shedding was completely inhibited by scavengers of ROS, whereas calcium-stimulated shedding was partially inhibited by ROS scavenging. Hydrogen peroxide restored TGF-alpha shedding after calcium chelation. Importantly, we also found that ATP-induced shedding was independent of the cytoplasmic NADPH oxidase complex. Instead, mitochondrial ROS production increased in response to ATP and mitochondrial oxidative complex activity was required to activate TACE-dependent shedding. These results reveal an essential role for mitochondrial ROS in regulating GPCR-induced growth factor shedding.

Insulin and epidermal growth factor suppress basal glucose-6-phosphatase catalytic subunit gene transcription through overlapping but distinct mechanisms.

The G6Pase (glucose-6-phosphatase catalytic subunit) catalyses the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate to glucose. We show here that, in HepG2 hepatoma cells, EGF (epidermal growth factor) inhibits basal mouse G6Pase fusion gene transcription. Several studies have shown that insulin represses basal mouse G6Pase fusion gene transcription through FOXO1 (forkhead box O1), but Stoffel and colleagues have recently suggested that insulin can also regulate gene transcription through FOXA2 (forkhead box A2) [Wolfrum, Asilmaz, Luca, Friedman and Stoffel (2003) Proc. Natl. Acad. Sci. 100, 11624-11629]. A combined GR (glucocorticoid receptor)-FOXA2 binding site is located between -185 and -174 in the mouse G6Pase promoter overlapping two FOXO1 binding sites located between (-188 and -182) and (-174 and -168). Selective mutation of the FOXO1 binding sites reduced the effect of insulin, whereas mutation of the GR/FOXA2 binding site had no effect on the insulin response. In contrast, selective mutation of the FOXO1 and GR/FOXA2 binding sites both reduced the effect of EGF. The effect of these mutations was additive, since the combined mutation of both FOXO1 and GR/FOXA2 binding sites reduced the effect of EGF to a greater extent than the individual mutations. These results suggest that, in HepG2 cells, GR and/or FOXA2 are required for the inhibition of basal G6Pase gene transcription by EGF but not insulin. EGF also inhibits hepatic G6Pase gene expression in vivo, but in cultured hepatocytes EGF has the opposite effect of stimulating expression, an observation that may be explained by a switch in ErbB receptor sub-type expression following hepatocyte isolation.

Cultured rat hepatocytes upregulate Akt and ERK in an ErbB-2-dependent manner.

Epidermal growth factor (EGF) stimulates freshly plated adult hepatocytes to synthesize DNA, but only after they pass through a lag phase of 40 h following EGF exposure. The longer the cells are maintained, they become more responsive to EGF and the lag phase shortens. Maximal EGF-mediated stimulation of DNA synthesis requires the induction of ErbB2, which is not normally expressed in adult hepatocytes. We used immunological methods to demonstrate increased expression during culture of two gene families required for EGF to stimulate hepatocyte DNA synthesis: Akt and ERK 1/2. Both families showed hyperexpression in culture particularly when cells were exposed to insulin and EGF. Unlike CDK-2 and cyclin D1, integral mediators of the G1/S phase transition, ERK 1/2 and Akt appeared in the absence of EGF, particularly when insulin was present. This hyperexpression, which high concentrations of dexamethasone reversed, increased basal and growth factor-stimulated phosphorylation of Akt and ERK 1/2. Pharmacological blockade of phosphatidylinositol kinase suppressed the Akt increase whereas pharmacological blockade or small interfering RNA downregulation of ErbB2 inhibited both Akt and ERK 1/2 expression. All three Akt isoforms contributed to the increase in total Akt. EGF but not insulin specifically upregulated Akt 2 and 3. Since Akt and ERK 1/2 are also hyperexpressed in poorly differentiated hepatomas, their dysregulation in cancer may involve transcriptional mechanisms normally operative in cultured hepatocytes. We hypothesize that the induction and activation of ErbB2 increases the expression of these kinases, enhancing the responsiveness of hepatocytes to EGF as they adapt to culture.

Evidence of metabolic syndrome in lean children with premature pubarche at diagnosis.

We investigated for evidence of early metabolic syndrome irrespective of body mass index (BMI) in subjects with premature pubarche (PP). Ten children with PP were compared with controls matched for age, sex, ethnicity, and BMI. Congenital adrenal hyperplasia and other known causes of PP were excluded by standard methods. Anthropometry, blood pressure (BP), dual-energy x-ray absorptiometry body scan, fasting blood lipid profile, and cytokines were obtained. The children were divided into 2 groups: (1) the total group of children with PP, and their age-, sex-, ethnicity-, and BMI-matched controls and (2) those with PP and normal BMI (<19 kg/m(2)) and their matched controls selected from the original groups. The PP subjects with normal BMI (S(1)) showed significantly higher systolic BP (P = .028), diastolic BP (P = .028), and mean arterial pressure (P = .018) compared with matched controls (C(1)). Nevertheless, for both groups, all the above parameters were statistically not significant when corrected for height. Fat distribution in PP subjects indicated significantly higher android (P = .047) and android-gynoid ratio (P = .013). Normal-BMI PP children had significantly higher android-gynoid ratio fat distribution compared with their matched controls (P = .037). Trunk fat percentage (p: 0.04) and trunk fat (grams) (P = .007) were significantly elevated in PP children compared with matched controls. Again, for both groups, all the above parameters were not statistically significant when corrected for height. The PP subjects had significantly higher tumor necrosis factor (TNF)-alpha (P = .038) and interleukin-8 (picograms per milliliter) (P = .05) compared with matched controls. Normal-BMI PP children also had higher TNF-alpha (P = .028) compared with matched controls. When corrected for height, TNF-alpha was higher in the total (P = .037) and normal-BMI (P = .043) PP children. Premature pubarche can be linked to markers of the metabolic syndrome in lean children.

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes.

Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10(-6) M) on DNA synthesis. When we added transforming growth factor-alpha at the time of plating, 10(-6) M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24-48 h after plating, 10(-6) M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10(-8) M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10(-6) M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling.

The proliferative effects of EGF in liver have been extensively investigated in cultured hepatocytes. We studied the effects of EGF, insulin, and other growth regulators on the expression, interaction, and signaling of ErbB receptors in primary cultures of adult rat hepatocytes. Using immunological methods and ErbB tyrosine kinase inhibitors, we analyzed the expression and signaling patterns of the ErbB kinases over 120 h of culture. Basal and EGF-stimulated protein tyrosine phosphorylation increased as cells adapted in vitro. EGF receptor (EGFr) expression declined in the first 24 h, whereas ErbB3 expression rose. Although ErbB2 was not present in freshly isolated hepatocytes, EGF and insulin independently induced ErbB2 while suppressing ErbB3 expression. Low concentrations of EGF and insulin synergistically stimulated ErbB2 expression and DNA synthesis. The greatest increase in ErbB2, which is normally expressed by fetal and neonatal hepatocytes, occurred shortly before the onset of DNA synthesis (> 40 h). EGF promoted EGFr and ErbB2 coassociation, stimulating tyrosine phosphorylation of both proteins. In contrast, heregulin beta1 (HRG-beta1) did not promote ErbB2 and ErbB3 coassociation. A selective tyrphostin inhibitor of ErbB2 suppressed EGF-stimulated DNA synthesis, but maximum suppression required the blockade of the EGFr kinase as well. Maximal EGF stimulation of DNA synthesis in vitro depends on the induction of ErbB2 and involves an EGFr-ErbB2 heterodimer. The ability of insulin to induce ErbB2 suggests both a mechanism for the synergy between insulin and EGF and a possible metabolic control of ErbB2 in vivo.

Inactivation of TGF-beta signaling in hepatocytes results in an increased proliferative response after partial hepatectomy.

The transforming growth factor beta (TGF-beta) signaling pathway, which is activated by the TGF-beta receptor complex consisting of type I and type II TGF-beta receptors (TGFBR1 and TGFBR2), regulates cell growth and death. TGF-beta and components of its signaling pathway, particularly TGFBR2, have been implicated as tumor suppressor genes and important antimitogenic factors in the gastrointestinal tract and liver. An in vivo approach to study these effects has been hindered by the embryonic lethality of Tgfbr2(-/-) mice and poor viability of the Tgfb1(-/-) mice. Consequently, we have developed a hepatocyte-specific Tgfbr2 knockout mouse, the Alb-cre Tgfbr2(flx/flx) mouse, to study the physiologically relevant effects of TGF-beta signaling on epithelial cell proliferation in vivo. After 70% hepatectomy, we observed increased proliferation and an increased liver mass : body weight ratio in the Alb-cre Tgfbr2(flx/flx) mice compared to Tgfbr2(flx/flx) mice. We also observed decreased expression and increased phosphorylation of p130 in the livers from the Alb-cre Tgfbr2(flx/flx) mice as well as increased expression of cyclin E, which is transcriptionally regulated, in part, by p130:E2F4. Consistent with these results, in a hepatocyte cell line derived from the Tgfbr2(flx/flx) mice, we found that TGF-beta increases the nuclear localization of E2F4, and presumably the transcriptional repression of the p130:E2F4 complex. Thus, we have demonstrated that TGF-beta signaling in vivo regulates the mitogenic response in the regenerating liver, affecting the liver mass : body weight ratio after partial hepatectomy, and that these mitogenic responses are accompanied by alterations in p130 expression and phosphorylation, implicating p130 as one of the proteins regulated in vivo by TGF-beta during liver regeneration.

Selective roles for tumor necrosis factor alpha-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family: the juxtamembrane stalk determines cleavage efficiency.

Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence.

TACE/ADAM17 processing of EGFR ligands indicates a role as a physiological convertase.

EGF family growth factors, including transforming growth factor-alpha (TGFalpha), amphiregulin (AR), and heparin-binding EGF (HB-EGF), are invariably expressed as transmembrane precursors that are cleaved at one or more sites in the extracellular domain to release soluble growth factor. Considerable attention has focused on the identification of proteases responsible for these processing events. We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the generation of soluble TGFalpha from its transmembrane precursor, proTGFalpha. Here, we review our findings that primary keratinocytes from Tace(deltaZn/deltaZn) mice, which express a nonfunctional TACE, released dramatically lower levels of soluble TGFalpha compared to their normal counterparts, even though TGFalpha mRNA and cell-associated protein levels were similar in the two cell populations. Restoration of TACE activity in Tace(deltaZn/deltaZn) cells increased shedding of TGFalpha species, including the mature, 6-kDa protein. Further, exogenous TACE enzyme accurately cleaved the N-terminal processing site of proTGFalpha in cell lysates, as well as both physiologic sites of a soluble proTGFalpha ectodomain. TACE also accurately cleaved peptide substrates corresponding to the processing sites of several additional EGF family members, and restoration of TACE activity enhanced the shedding of soluble AR and HB-EGF proteins from Tace(deltaZn/deltaZn) cells. Finally, reduction of functional TACE gene dosage greatly exacerbated the open-eye defect of Egfr(wa-2/wa-2) newborns, which is regulated by redundant actions of several EGF family ligands. The implications of these results for the biology of the EGF family and TACE are discussed.

Diverse expression of ErbB receptor proteins during rat liver development and regeneration.

BACKGROUND & AIMS: The protein expression and interactions of the ErbB receptors were examined in different liver proliferation models in vivo and in vitro, including ontogeny and regeneration following partial hepatectomy. METHODS: Expression and tyrosine phosphorylation status of specific ErbB proteins were studied by immunologic methods. RESULTS: The epidermal growth factor receptor, ErbB2, and ErbB3 were the only ErbB proteins detected in the liver parenchyma on embryonic day 19. ErbB2 disappeared by the third week after birth and could not be appreciably induced in the adult animal by partial hepatectomy. ErbB2 was also detected in multipotent stem (RLE) and hepatoma (H4IIe) cell lines as well as in fetal, but not adult, hepatocyte cultures. Only epidermal growth factor receptor and ErbB3 were detected in adult liver, and both showed circadian variation in protein expression. ErbB4 was not detected in any model. Patterns of ligand-induced ErbB phosphorylation differed between fetal and adult hepatocytes. CONCLUSIONS: Complex and independent programs regulate the ErbB receptors, with implications for differential cell signaling in hepatic development and regeneration.

Premature pubarche in girls is associated with functional adrenal but not ovarian hyperandrogenism.

OBJECTIVE: We hypothesized that there would be evidence of functional ovarian hyperandrogenism in girls with premature pubarche (PP) at diagnosis. METHODS: White girls <8 years of age and black girls <6 years with PP (n = 15) were studied. Prepubertal girls (n = 13; 5.3-10.9 years) and early pubertal girls (n = 8) served as control subjects. The biochemical marker for functional ovarian hyperandrogenism was the 17-hydroxyprogesterone (17-OHP), androstenedione (AD), and estradiol (E2) response to subcutaneous leuprolide during adrenal suppression with dexamethasone. This was studied in girls with PP and in control subjects. RESULTS: ACTH stimulated 17-hydroxypregnenolone (17-OH Preg), dehydroepiandrosterone (DHEA), and AD levels, and 17-OH Preg:17-OHP and DHEA:AD ratios were significantly higher in girls with PP than in prepubertal control subjects (n = 18) (P < or =.003). The ovarian response to leuprolide stimulation was comparable in girls with PP and prepubertal control subjects, but the response in prepubertal study subjects was significantly lower than in pubertal control subjects (P =.016 for Delta17-OHP, P =.001 for DeltaAD, and P =.026 for DeltaE2). CONCLUSIONS: Contrary to the hypothesis, PP in girls was not associated with prepubertal evidence of ovarian hyperandrogenism but was associated with functional adrenal hyperandrogenism.

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability.

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.