Inflation of 430-parsec bipolar radio bubbles in the Galactic Centre by an energetic event.

The Galactic Centre contains a supermassive black hole with a mass of four million Suns1 within an environment that differs markedly from that of the Galactic disk. Although the black hole is essentially quiescent in the broader context of active galactic nuclei, X-ray observations have provided evidence for energetic outbursts from its surroundings2. Also, although the levels of star formation in the Galactic Centre have been approximately constant over the past few hundred million years, there is evidence of increased short-duration bursts3, strongly influenced by the interaction of the black hole with the enhanced gas density present within the ring-like central molecular zone4 at Galactic longitude |l| < 0.7 degrees and latitude |b| < 0.2 degrees. The inner 200-parsec region is characterized by large amounts of warm molecular gas5, a high cosmic-ray ionization rate6, unusual gas chemistry, enhanced synchrotron emission7,8, and a multitude of radio-emitting magnetized filaments9, the origin of which has not been established. Here we report radio imaging that reveals a bipolar bubble structure, with an overall span of 1 degree by 3 degrees (140 parsecs × 430 parsecs), extending above and below the Galactic plane and apparently associated with the Galactic Centre. The structure is edge-brightened and bounded, with symmetry implying creation by an energetic event in the Galactic Centre. We estimate the age of the bubbles to be a few million years, with a total energy of 7 × 1052 ergs. We postulate that the progenitor event was a major contributor to the increased cosmic-ray density in the Galactic Centre, and is in turn the principal source of the relativistic particles required to power the synchrotron emission of the radio filaments within and in the vicinity of the bubble cavities.

A comprehensive analysis of the CDKN2A gene in childhood acute lymphoblastic leukemia reveals genomic deletion, copy number neutral loss of heterozygosity, and association with specific cytogenetic subgroups.

Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. We assessed the principal mode of inactivation in childhood acute lymphoblastic leukemia (ALL) and frequency in biologically relevant subgroups. Mutation or methylation was rare, whereas genomic deletion occurred in 21% of B-cell precursor ALL and 50% of T-ALL patients. Single nucleotide polymorphism arrays revealed copy number neutral (CNN) loss of heterozygosity (LOH) in 8% of patients. Array-based comparative genomic hybridization demonstrated that the mean size of deletions was 14.8 Mb and biallelic deletions composed a large and small deletion (mean sizes, 23.3 Mb and 1.4 Mb). Among 86 patients, only 2 small deletions were below the resolution of detection by fluorescence in situ hybridization. Patients with high hyperdiploidy, ETV6-RUNX1, or 11q23/MLL rearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1 rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2A deletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2A deletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2A inactivation suggests the presence of other relevant genes in this region.

Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder.

The epidermal growth factor receptor (HER1) has been implicated in regenerative growth and proliferative diseases of the human bladder epithelium (urothelium), however a cognate HER1 ligand that can act as a growth factor for normal human urothelial cells (HUC) has not been identified. Here we show that heparin-binding EGF-like growth factor (HB-EGF), an activating HER1 ligand, is an autocrine regulator of HUC growth. This conclusion is based on demonstration of HB-EGF synthesis and secretion by primary culture HUC, identification of HER1 as an activatable HB-EGF receptor on HUC surfaces, stimulation of HUC clonal growth by HB-EGF, inhibition of HB-EGF-stimulated growth by heparin and of log-phase growth by CRM 197, a specific inhibitor of HB-EGF/HER1 interaction, and identification of human urothelium as a site of HB-EGF precursor (proHB-EGF) synthesis in vivo. ProHB-EGF expression was also detected in the vascular and detrusor smooth muscle of the human bladder. These data suggest a physiologic role for HB-EGF in the regulation of urothelial proliferation and regeneration subsequent to mucosal injury. Expression of proHB-EGF is also a feature of differentiated vascular and detrusor smooth muscle in the bladder. Because proHB-EGF is known to be the high affinity diphtheria toxin (DT) receptor in human cells, synthesis of the HB-EGF precursor by human urothelium also suggests the possibility of using the DT-binding sites of proHB-EGF as an in vivo target for the intraluminal treatment of urothelial diseases.

The phosphatidylinositol 3'-kinase pathway is a dominant growth factor-activated cell survival pathway in LNCaP human prostate carcinoma cells.

Intracellular signaling pathways that mediate survival of prostate carcinoma (PCa) cells are poorly understood. We examined the potential role of the phosphatidylinositol 3' kinase (PI3K) pathway as a mediator of cell survival in LNCaP human PCa cells, which express a variety of properties characteristic of human prostate cancer. LNCaP cell cultures rapidly became apoptotic when treated with the specific PI3K inhibitors, wortmannin and LY294002. In contrast, apoptosis was not induced when the cells were treated with: (a) rapamycin, an inhibitor of the ribosomal S6 kinase pp70S6K, which acts downstream of PI3K; (b) PD98059, a specific inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) kinase (MEK); or (c) the antiandrogen, Casodex; or when the cells were cultured under androgen-depleted conditions. Apoptosis induced by PI3K inhibition was attenuated by: (a) dihydrotestosterone; or (b) the ErbB1 activating ligands [epidermal growth factor (EGF), transforming growth factor alpha, or heparin-binding EGF-like growth factor]. In response to ErbB1 activation by ligand, the p85 regulatory subunit of PI3K associated specifically with ErbB3 but not detectably with ErbB1. The anti-apoptotic effect of ErbB1 activation was significantly reduced when cells were treated simultaneously with wortmannin and PD98059. These data indicate that survival signals can be evoked in LNCaP cells by several distinct pathways and can be triggered by nuclear and cell-surface receptors. Constitutive signaling through the PI3K pathway is required to prevent cell death in LNCaP, whereas activation of the Erk/MAPK and androgen response pathways is not obligatory for cell survival. These results also show that survival signals, as distinguished from mitogenic signals, can be evoked in PCa cells by ErbB1 ligands known to be synthesized within the human prostate.

Long-term right ventricular implantable cardioverter-defibrillator lead performance in arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive disease characterized by replacement of normal myocardium by fibrofatty tissue. The right ventricular (RV) apex is the typical target for implantable cardioverter-defibrillator (ICD) lead placement, raising concerns for suboptimal lead performance in medium- to long-term follow-up. OBJECTIVE: The purpose of this study was to determine whether placement of ICD leads at the RV apex was associated with performance deterioration of medium-term leads in ARVC patients compared to non-ARVC patients. METHODS: In this multicenter, retrospective, case-control study, ICD lead performance measures of R-wave, impedance, and pacing thresholds were compared at baseline and between 1-year and 5-year postimplantation follow-up using mixed-effect models adjusted for age and sex. RESULTS: One hundred one ARVC patients (49 women, age 50.6 ± 14.5 years) were compared to 56 control patients (37 women, age 48.2 ± 14.2 years). The mean difference in R wave between years 1 and 2 was -0.85 mV (P = .16) compared to a mean difference at years 5 and 6 of -1.85 mV (P = .02). There was no difference in impedance or pacing threshold or in lead lifetime between the 2 groups over 6-year follow-up (5.91 ± 3.89 years vs 5.48 ± 3.70 years, P = .239). CONCLUSION: In ARVC patients with ICD leads implanted in the RV apex, ventricular sensing deteriorates significantly during medium-term follow-up. Septal RV lead placement should be explored as the first choice at implantation.

Outcome of Apparently Unexplained Cardiac Arrest: Results From Investigation and Follow-Up of the Prospective Cardiac Arrest Survivors With Preserved Ejection Fraction Registry.

BACKGROUND: The Cardiac Arrest Survivors with Preserved Ejection Fraction Registry (CASPER) enrolls patients with apparently unexplained cardiac arrest and no evident cardiac disease to identify the pathogenesis of cardiac arrest through systematic clinical testing. Exercise testing, drug provocation, advanced cardiac imaging, and genetic testing may be useful when a cause is not apparent. METHODS AND RESULTS: The first 200 survivors of unexplained cardiac arrest from 14 centers across Canada were evaluated to determine the results of investigation and follow-up (age, 48.6±14.7 years, 41% female). Patients were free of evidence of coronary artery disease, left ventricular dysfunction, or evident repolarization syndromes. Advanced testing determined a diagnosis in 34% of patients at baseline, with a diagnosis emerging during follow-up in 7% of patients. Of those who were diagnosed, 28 (35%) had an underlying structural condition and 53 (65%) had a primary electric disease. During a mean follow-up of 3.15±2.34 years, 23% of patients had either a shock or an appropriate antitachycardia pacing from their implantable cardioverter defibrillator, or both. The implantable cardioverter defibrillator appropriate intervention rate was 8.4% at 1 year and 18.1% at 3 years, with no clear difference between diagnosed and undiagnosed subjects, or between those diagnosed with a primary electric versus structural pathogenesis. CONCLUSIONS: Obtaining a diagnosis in previously unexplained cardiac arrest patients requires systematic clinical testing and regular follow-up to unmask the cause. Nearly half of apparently unexplained cardiac arrest patients ultimately received a diagnosis, allowing for improved treatment and family screening. A substantial proportion of patients received appropriate implantable cardioverter defibrillator therapy during medium-term follow-up. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00292032.

Amphiregulin is coordinately expressed with heparin-binding epidermal growth factor-like growth factor in the interstitial smooth muscle of the human prostate.

Peptide growth factors have been proposed as mediators of smooth muscle-epithelial cell interactions in the human prostate; however, the identity of these molecules has not been established. In this study, we compared expression levels of messenger RNAs (mRNAs) encoding the epidermal growth factor (EGF) receptor-related receptor tyrosine kinases (ErbB1 through 4), the six EGF receptor ligands, EGF, transforming growth factor (TGF)-alpha, amphiregulin (ARG), HB-EGF, betacellulin, and epiregulin, and the related molecule heregulin-alpha, in a series of 10 prostate tissue specimens. Only EGF showed a disease-specific association, with increased mRNA levels in four of five PCa specimens in comparison to matched normal tissue from the same subject. In contrast, ARG and HB-EGF mRNAs showed a coordinate pattern of expression in 7/10 specimens that was distinct from all other growth factor or receptor genes examined and from mRNAs for prostate specific antigen, the androgen receptor and GAPDH, a house-keeping enzyme. Analysis of an additional series of benign prostatic hyperplasia and prostate cancer specimens from 60 individuals confirmed that ARG and HB-EGF mRNA levels varied in a highly coordinate manner (r = 0.93; P < 0.0001) but showed no association with disease. ARG was immunolocalized largely to interstitial smooth muscle cells (SMC), previously identified as the site of synthesis of HB-EGF in the prostate, while the cognate ARG and HB-EGF receptor, ErbB1, was localized exclusively to ductal epithelial cells and carcinoma cells. Although ARG was a relatively poor mitogen for Balb/c3T3 cells in comparison to HB-EGF, it was similar in potency to HB-EGF in stimulating human prostate epithelial cell growth, suggesting that prostate epithelia may be a physiologic target for ARG in vivo. Expression of both ARG and HB-EGF mRNAs was induced in cultured prostate SMC by fibroblast growth factor-2, a human prostate SMC mitogen linked to prostate disease. These findings indicate that ARG and HB-EGF are likely to be key mediators of directional signaling between SMC and epithelial cells in the human prostate and appear to be coordinately regulated.

Mechanical strain delivers anti-apoptotic and proliferative signals to gingival fibroblasts.

Physical forces play a critical role in the survival and proliferation of many cell types, including fibroblasts. Gingival fibroblasts are exposed to mechanical stress during mastication, orthodontic tooth movement, and wound healing following periodontal surgery. The aim of this study was to examine the effect of mechanical strain on human gingival fibroblasts (hGF). Cells were subjected to short-term (up to 60 min) and long-term (up to 48 hrs) 20% average elongation at 0.1 Hz. We monitored survival signaling by evaluating the phosphorylation status and localization of Forkhead box (FoxO) family members, which are mediators of apoptosis. We also examined strain-induced proliferation by measuring the level of proliferating cell nuclear antigen (PCNA). We observed that cyclic strain caused the phosphorylation and retention in the cytoplasm of FoxO family members. Moreover, mechanical strain resulted in increased ERK kinase phosphorylation and PCNA expression. In conclusion, cyclic strain delivers anti-apoptotic and proliferative stimuli to hGF.

Scaffold attachment factor B1 regulates the androgen receptor in concert with the growth inhibitory kinase MST1 and the methyltransferase EZH2.

The androgen receptor (AR) is a transcription factor that employs many diverse interactions with coregulatory proteins in normal physiology and in prostate cancer (PCa). The AR mediates cellular responses in association with chromatin complexes and kinase cascades. Here we report that the nuclear matrix protein, scaffold attachment factor B1 (SAFB1), regulates AR activity and AR levels in a manner that suggests its involvement in PCa. SAFB1 mRNA expression was lower in PCa in comparison with normal prostate tissue in a majority of publicly available RNA expression data sets. SAFB1 protein levels were also reduced with disease progression in a cohort of human PCa that included metastatic tumors. SAFB1 bound to AR and was phosphorylated by the MST1 (Hippo homolog) serine-threonine kinase, previously shown to be an AR repressor, and MST1 localization to AR-dependent promoters was inhibited by SAFB1 depletion. Knockdown of SAFB1 in androgen-dependent LNCaP PCa cells increased AR and prostate-specific antigen (PSA) levels, stimulated growth of cultured cells and subcutaneous xenografts and promoted a more aggressive phenotype, consistent with a repressive AR regulatory function. SAFB1 formed a complex with the histone methyltransferase EZH2 at AR-interacting chromatin sites in association with other polycomb repressive complex 2 (PRC2) proteins. We conclude that SAFB1 acts as a novel AR co-regulator at gene loci where signals from the MST1/Hippo and EZH2 pathways converge.

Molecular cytogenetic characterization of TCF3 (E2A)/19p13.3 rearrangements in B-cell precursor acute lymphoblastic leukemia.

The t(1;19)(q23;p13.3) is one of the most common chromosomal abnormalities in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and usually gives rise to the TCF3-PBX1 fusion gene. Additional rare, and sometimes cytogenetically cryptic, translocations involving the TCF3 gene have also been described. Using a dual color split-signal fluorescence in situ hybridization (FISH) probe, we have investigated the involvement of this gene in a series of BCP-ALLs harboring 19p13 translocations, as well as an unselected patient cohort. The TCF3 gene was shown to be involved in the majority of cases with a cytogenetically visible t(1;19) translocation, while the remaining TCF3-negative ALLs demonstrated breakpoint heterogeneity. Although most "other" 19p13 translocations did not produce a split-signal FISH pattern, a novel t(13;19)(q14;p13) involving TCF3 was discovered. A prospective screen of 161 children with BCP-ALL revealed a cryptic t(12;19)(p13;p13), another novel TCF3 rearrangement, and a series of patients with submicroscopic deletions of TCF3. These results demonstrate the utility of a split-signal FISH strategy in confirming the involvement of the TCF3 gene in 19p13 rearrangements and in identifying novel and cryptic TCF3 translocations. In addition to its role as a fusion partner gene, we propose that TCF3 can also act as a tumor suppressor gene in BCP-ALL.

Clinics in general practice. A case for the gynaecologist?

PIP: The case of a 31-year-old female patient with 2 children who was sterilized 7 years ago at age 24 years and has a 3-year history of low bilateral abdominal pain is discussed. The patient was investigated at the surgical outpatient department and has been recommended to a gynecologist. The symptoms lack suggestion of gynecological disease. Some of the questions to be asked are why she was sterilized, why not her husband, and what method was used. Were there any complications in sterilization operations 7 years ago that resulted in abdominal pain? Whoever takes on the case should question what the quality of the woman's life was before the operation and how it has since changed. It would be helpful to know if she would have liked a 3rd child and if her sexual feelings have changed. At the time she was sterilized, it was unusual for a woman to be sterilized at the age of 24 years with 2 children. There were probably strong medical or psychiatric indications then. One suspects either tubal disease after sterilization or chronic pelvic inflammatory disease as the likely diagnosis. If it becomes apparent that she wants another baby perhaps tubal reanastomosis could be accomplished.^ieng

Induction of anchorage-independent growth by amphiregulin.

We have previously shown that the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AR) exhibits low potency as a result of its C-terminal truncation. This led us to investigate whether its inability to promote anchorage-independent growth (AIG) of normal cells arose because of its compromised interaction with EGFR. Wild type AR(1-84) was tested in AIG and mitogenesis assays using NRK-49F or NR6/HER fibroblasts. In contrast to NR6/HER cells, the response of NRK-49F fibroblasts to AR was much lower than expected. As the effect of AR was heparin-insensitive, contributions from heparan sulphate proteoglycan interactions could not explain the differing sensitivities of the cells. Comparison of the effects of AR on two additional cell lines indicated that low EGFR number correlated with AR insensitivity: this suggested that the low potency of AR precluded activation of sufficient receptors to elicit a response. Consistent with this proposal, a modified form of AR (AR[1-90(leu86)]) with enhanced potency was able to induce AIG of NRK-49F fibroblasts. Thus, the ability of AR to promote AIG is determined both by ligand potency and the EGFR complement of cells.

Heparin-binding epidermal growth factor-like growth factor is an autocrine mediator of human prostate stromal cell growth in vitro.

PURPOSE: The physiological mechanisms by which soluble mediators of cell proliferation and survival alter expansion of the prostatic stroma in benign prostatic hyperplasia are poorly understood. We recently identified heparin-binding epidermal growth factor like growth factor (HB-EGF) as a product predominantly of the smooth muscle cell compartments of the adult human prostate. We assess the potential role of this growth factor as a stromal cell regulator. MATERIALS AND METHODS: Primary cultures of desmin and alpha-actin positive human prostate stromal cells were shown to express several cell associated HB-EGF isoforms as well as the primary cognate HB-EGF receptor, ErbB1/HER1, suggesting the existence of an autocrine or juxtacrine regulatory loop. The related receptor tyrosine kinase, ErbB2/HER2, was also expressed as assessed by reverse transcriptase (RT) polymerase chain reaction (PCR). HB-EGF messenger RNA levels in human prostate stromal cells increased modestly (70%) in response to a repetitive mechanical stimulus, a lower response than has been reported for neonatal rat bladder smooth muscle cells, in which HB-EGF was originally identified as a mechanically responsive gene. RESULTS: HB-EGF, epidermal growth factor and basic fibroblast growth factor stimulated human prostate stromal cell growth, while a specific antagonist of HB-EGF, [Glu52]-diphtheria toxin/CRM197, inhibited human prostate stromal growth in serum-free medium by a mechanism that did not involve increased apoptosis. A function blocking antibody against CD9/DRAP27/MRP-1, a cell surface binding partner of the membrane form of HB-EGF, also stimulated human prostate stromal cell proliferation. CONCLUSIONS: HB-EGF is an endogenously produced human prostate stromal cell growth factor and, thus, may have a role as a physiologically relevant autocrine or juxtacrine mediator of stromal expansion in benign prostatic hyperplasia.

Plasma levels of vascular endothelial growth factor are increased in patients with metastatic prostate cancer.

OBJECTIVES: Vascular endothelial growth factor (VEGF) is a cytokine that plays an important role in tumor angiogenesis. VEGF is overexpressed in many human cancers, including prostate cancer, but circulating levels of VEGF in patients with prostate cancer have not been reported. In this study, we analyzed plasma concentrations of VEGF in a cohort of patients with prostate cancer and compared them with a normal population. METHODS: Twenty-six healthy, cancer-free individuals and 80 patients with prostate cancer (54 patients with localized prostate cancer and 26 patients with metastatic prostate cancer [bone or lymph node positive]) were analyzed in this study. Blood was drawn in the same fashion from all individuals and deposited in tubes containing ethylenediaminetetraacetic acid as anticoagulant. Plasma was extracted and VEGF concentrations were determined using a quantitative sandwich enzyme immunoassay technique. RESULTS: Median plasma VEGF was 28.5 pg/mL (interquartile range 19.3 to 57.0) in patients with metastases; 7.0 pg/mL (interquartile range 0 to 26.5) in patients with localized disease, and 0 pg/mL (interquartile range 0 to 24) in controls. These differences were statistically significant (P <0.001). When compared group by group, the metastatic group had significantly higher plasma VEGF than the localized disease group and the control group (P = 0.003 and P <0.001, respectively). There was a tendency for plasma VEGF to be higher in the localized disease group than in the control group, a trend that almost reached statistical significance (P = 0.038). Using a cutoff of 18 pg/mL, the sensitivity and specificity of the test in differentiating between patients with and without metastatic disease was 81% and 71%, respectively. The odds of metastatic disease were almost 10 times greater for patients with VEGF values greater than 18 pg/mL than for those with values less than 18 pg/mL. There was no correlation between age and plasma VEGF values or between plasma VEGF and serum prostate-specific antigen (PSA). However, patients with serum PSA greater than 20 ng/mL had significantly higher plasma VEGF values than patients with serum PSA less than 20 ng/mL (P <0.001). No direct relation was found between Gleason sum and plasma VEGF, although VEGF levels were higher in patients with Gleason sums of 8 to 10 than in patients with lower Gleason sums. CONCLUSIONS: Our study indicates that patients with metastatic prostate cancer have higher plasma VEGF levels than patients with localized disease or healthy controls. A larger prospective study is needed to confirm the predictive utility of VEGF.

Cyclic stretch activates p38 SAPK2-, ErbB2-, and AT1-dependent signaling in bladder smooth muscle cells.

Cyclic mechanical stretch of bladder smooth muscle cells (SMC) increases rates of DNA synthesis and stimulates transcription of the gene for heparin-binding epidermal growth factor-like growth factor (HB-EGF), an ErbB1/EGF receptor ligand that has been linked to hypertrophic bladder growth. In this study we sought to clarify the signaling pathways responsible for mechanotransduction of the stretch stimulus. HB-EGF mRNA levels, DNA synthesis, and AP-1/Ets DNA binding activities were induced by repetitive stretch of primary culture rat bladder SMC. Inhibitors of the p38 SAPK2 pathway, the angiotensin receptor type 1 (AT1), and the ErbB2 tyrosine kinase reduced each of these activities, while an inhibitor of the extracellular signal-regulated kinase mitogen-activated protein kinase (Erk-MAPK) pathway had no effect. Stretch rapidly activated stress-activated protein kinase 2 (p38 SAPK2) and Jun NH(2)-terminal kinase (JNK)/SAPK pathways but not the Erk-MAPK pathway and induced ErbB2 but not ErbB1 phosphorylation. Angiotensin II (ANG II) a bladder SMC mitogen previously linked to the stretch response, did not activate ErbB2, and ErbB2 activation occurred in response to stretch in the presence of an ANG receptor inhibitor, indicating that activation of the AT1-mediated pathway and the ErbB2-dependent pathway occurs by independent mechanisms. p38 SAPK2 and JNK/SAPK signaling also appeared to be independent of the ErbB2 and AT1 pathways. These findings indicate that stretch-stimulated DNA synthesis and gene expression in normal bladder SMC occur via multiple independent receptor systems (e.g., AT1 and ErbB2) and at least one MAPK pathway (p38 SAPK2). Further, we show that the Erk-MAPK pathway, which in most systems is linked to receptor-dependent cell growth responses, is not involved in progression to DNA synthesis or in the response of the HB-EGF gene to mechanical forces.

Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C.

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca2+ ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC.