William E. Russell

An Essential Role for Ectodomain Shedding in Mammalian Development

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

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

We previously implicated tumor necrosis factor-α converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-α (TGF-α), pro-TGF-α. Here we examined TGF-α processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-α as compared with wild-type cultures and that TGF-α cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-α and TACE cDNAs increased shedding of mature TGF-α with concomitant conversion of cell-associated pro-TGF-α to a processed form. Purified TACE accurately cleaved pro-TGF-α in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-α 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 thanTace +/+ Egfr wa-2 / wa-2 counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.

Epidermal growth factor-related peptides and their relevance to gastrointestinal pathophysiology

Growth factor biology has been one of the most exciting areas of gastroenterological research in recent years. Although many fundamental questions about growth factors and their relevance to the gastrointestinal tract remain unanswered and unexplored, the available data point to major clinical significance in a large number of gastrointestinal disorders. This report reviews the biology and significance of the epidermal growth factor (EGF) family, with particular emphasis on understanding the integrated function of the family and the relationships between family members in the context of gastrointestinal physiology and pathophysiology.

Reversible G1 Arrest Induced by Inhibition of the Epidermal Growth Factor Receptor Tyrosine Kinase Requires Up-regulation of p27KIP1 Independent of MAPK Activity

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G1 to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1–10 μm AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1–1 μm AG-1517 abrogated 125I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 μm. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G1 phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G1 arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G1 arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G1. These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G1 arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.

TACE/ADAM17 Processing of EGFR Ligands Indicates a Role as a Physiological Convertase

Abstract: EGF family growth factors, including transforming growth factor‐alpha (TGFα), 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 TGFα from its transmembrane precursor, proTGFα. Here, we review our findings that primary keratinocytes from TaceΔZn/ΔZn mice, which express a nonfunctional TACE, released dramatically lower levels of soluble TGFα compared to their normal counterparts, even though TGFα mRNA and cell‐associated protein levels were similar in the two cell populations. Restoration of TACE activity in TaceΔZn/ΔZn cells increased shedding of TGFα species, including the mature, 6‐kDa protein. Further, exogenous TACE enzyme accurately cleaved the N‐terminal processing site of proTGFα in cell lysates, as well as both physiologic sites of a soluble proTGFα 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 TaceΔZn/ΔZn cells. Finally, reduction of functional TACE gene dosage greatly exacerbated the open‐eye defect of Egfrwa‐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.

Increased production of transforming growth factor α following acute gastric injury

Transforming growth factor alpha (TGF-alpha) production recently has been found in normal mammalian gastric mucosa. Inasmuch as TGF-alpha and epidermal growth factor (EGF) both stimulate epithelial cell migration and proliferation and suppress gastric acid secretion, the authors of the current study proposed that these growth factors may participate in tissue repair after acute gastric mucosal injury. Consequently, TGF-alpha and EGF production were examined after orogastric administration of either acidified taurocholate or 0.6 mol/L HCl to rats. TGF-alpha messenger RNA (mRNA) expression increased in a dose- and time-dependent manner after administration of taurocholate, whereas EGF mRNA expression was not detected. Radioimmunoassay of gastric mucosal scrapings obtained 6 hours after gastric injury induced by 0.6 mol/L HCl showed a 2.1-fold increase in immunoreactive TGF-alpha but no increase in immunoreactive EGF. In addition, there was a 68-fold increase in immunoreactive TGF-alpha in gastric juice within 30 minutes of gastric instillation of HCl and, again, no increase in immunoreactive EGF. There is a rapid appearance of TGF-alpha in the gastric juice within 30 minutes of injury, which is followed by increased expression of TGF-alpha mRNA and protein in the gastric mucosa. These studies suggest that locally produced TGF-alpha may participate in gastric mucosal repair following acute gastric injury to rats.

Liver regeneration and hepatocarcinogenesis in transforming growth factor‐α‐targeted mice

Transforming growth factor‐α (TGFα), a member of the epidermal growth factor receptor ligand family, has been implicated in the regeneration and transformation of liver. Our recent development of mice that are homozygous for a disrupted TGFα gene allowed us to assess the requirement for this growth factor in these complex processes. We report here that although a 70% hepatectomy produced a significant increase in hepatic TGFα protein levels in wild‐type mice, liver regeneration nevertheless proceeded normally in the absence of the growth factor. The hepatocyte labeling indices determined for homozygous targeted and wild‐type mice at 36 and 48 h after hepatectomy were comparable, and the total liver DNA to body weight ratios 8 d after hepatectomy were essentially identical for the two genotypes. These results indicate that TGFα is not necessary for liver regeneration. To test its requirement in liver carcinogenesis, young mice were administered single doses of diethylnitrosamine (DEN) with or without subsequent chronic treatment with the promoting agent phenobarbital (PB). Both wild‐type and homozygous mutant male mice treated with DEN or DEN plus PB developed multiple preneoplastic foci or tumors by 9 mo of age with relatively high incidence. However, while five of 88 tumors in wild‐type mice attained a diameter greater than 5 mm and were classified as hepatocellular carcinomas, none of 132 tumors in livers of targeted mice reached this size. Furthermore, three of these large wild‐type tumors expressed significantly elevated levels of TGFα protein compared with normal liver. These results indicate that TGFα is not required for early events in chemically induced hepatocarcinogenesis but suggest that it could be important in the progression from small preneoplastic foci to large tumors.

Targeting of memory T cells with alefacept in new-onset type 1 diabetes (T1DAL study): 12 month results of a randomised, double-blind, placebo-controlled phase 2 trial

BACKGROUND Type 1 diabetes results from autoimmune targeting of the pancreatic β cells, likely mediated by effector memory T (Tem) cells. CD2, a T cell surface protein highly expressed on Tem cells, is targeted by the fusion protein alefacept, depleting Tem cells and central memory T (Tcm) cells. We postulated that alefacept would arrest autoimmunity and preserve residual β cells in patients newly diagnosed with type 1 diabetes. METHODS The T1DAL study is a phase 2, double-blind, placebo-controlled trial in patients with type 1 diabetes, aged 12-35 years who, within 100 days of diagnosis, were enrolled at 14 US sites. Patients were randomly assigned (2:1) to receive alefacept (two 12-week courses of 15 mg intramuscularly per week, separated by a 12-week pause) or a placebo. Randomisation was stratified by site, and was computer-generated with permuted blocks of three patients per block. All participants and site personnel were masked to treatment assignment. The primary endpoint was the change from baseline in mean 2 h C-peptide area under the curve (AUC) at 12 months. Secondary endpoints at 12 months were the change from baseline in the 4 h C-peptide AUC, insulin use, major hypoglycaemic events, and HbA1c concentrations. This trial is registered with ClinicalTrials.gov, number NCT00965458. FINDINGS Of 73 patients assessed for eligibility, 33 were randomly assigned to receive alefacept and 16 to receive placebo. The mean 2 h C-peptide AUC at 12 months increased by 0.015 nmol/L (95% CI -0.080 to 0.110) in the alefacept group and decreased by 0.115 nmol/L (-0.278 to 0.047) in the placebo group, and the difference between groups was not significant (p=0.065). However, key secondary endpoints were met: the mean 4 h C-peptide AUC was significantly higher (mean increase of 0.015 nmol/L [95% CI -0.076 to 0.106] vs decrease of -0.156 nmol/L [-0.305 to -0.006]; p=0.019), and daily insulin use (0.48 units per kg per day for placebo vs 0.36 units per kg per day for alefacept; p=0.02) and the rate of hypoglycaemic events (mean of 10.9 events per person per year for alefacept vs 17.3 events for placebo; p<0.0001) was significantly lower at 12 months in the alefacept group than in the placebo group. Mean HbA1c concentrations at week 52 were not different between treatment groups (p=0.75). So far, no serious adverse events were reported and all patients had at least one adverse event. In the alefacept group, 29 (88%) participants had an adverse event related to study drug versus 15 (94%) participants in the placebo group. In the alefacept group, 14 (42%) participants had grade 3 or 4 adverse events compared with nine (56%) participants in the placebo group; no deaths occurred. INTERPRETATION Although the primary outcome was not met, at 12 months, alefacept preserved the 4 h C-peptide AUC, lowered insulin use, and reduced hypoglycaemic events, suggesting efficacy. Safety and tolerability were similar in the alefacept and placebo groups. Alefacept could be useful to preserve β-cell function in patients with new-onset type 1 diabetes.Background Type 1 diabetes (T1D) results from autoimmune targeting of the pancreatic beta cells, likely mediated by effector memory T cells (Tems). CD2, a T cell surface protein highly expressed on Tems, is targeted by the fusion protein alefacept, depleting Tems and central memory T cells (Tcms). We hypothesized that alefacept would arrest autoimmunity and preserve residual beta cells in newly diagnosed T1D.

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

The transforming growth factor β (TGF-β) signaling pathway, which is activated by the TGF-β receptor complex consisting of type I and type II TGF-β receptors (TGFBR1 and TGFBR2), regulates cell growth and death. TGF-β 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 Tgfbr2flx/flx mouse, to study the physiologically relevant effects of TGF-β 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 Tgfbr2flx/flx mice compared to Tgfbr2flx/flx mice. We also observed decreased expression and increased phosphorylation of p130 in the livers from the Alb-cre Tgfbr2flx/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 Tgfbr2flx/flx mice, we found that TGF-β increases the nuclear localization of E2F4, and presumably the transcriptional repression of the p130:E2F4 complex. Thus, we have demonstrated that TGF-β 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-β during liver regeneration.

Costimulation Modulation With Abatacept in Patients With Recent-Onset Type 1 Diabetes: Follow-up 1 Year After Cessation of Treatment

OBJECTIVE We previously reported that 2 years of costimulation modulation with abatacept slowed decline of β-cell function in recent-onset type 1 diabetes (T1D). Subsequently, abatacept was discontinued and subjects were followed to determine whether there was persistence of effect. RESEARCH DESIGN AND METHODS Of 112 subjects (ages 6–36 years) with T1D, 77 received abatacept and 35 received placebo infusions intravenously for 27 infusions over 2 years. The primary outcome—baseline-adjusted geometric mean 2-h area under the curve (AUC) serum C-peptide during a mixed-meal tolerance test (MMTT) at 2 years—showed higher C-peptide with abatacept versus placebo. Subjects were followed an additional year, off treatment, with MMTTs performed at 30 and 36 months. RESULTS C-peptide AUC means, adjusted for age and baseline C-peptide, at 36 months were 0.217 nmol/L (95% CI 0.168–0.268) and 0.141 nmol/L (95% CI 0.071–0.215) for abatacept and placebo groups, respectively (P = 0.046). The C-peptide decline from baseline remained parallel with an estimated 9.5 months’ delay with abatacept. Moreover, HbA1c levels remained lower in the abatacept group than in the placebo group. The slightly lower (nonsignificant) mean total insulin dose among the abatacept group reported at 2 years was the same as the placebo group by 3 years. CONCLUSIONS Costimulation modulation with abatacept slowed decline of β-cell function and improved HbA1c in recent-onset T1D. The beneficial effect was sustained for at least 1 year after cessation of abatacept infusions or 3 years from T1D diagnosis.