The Role of The Role of CalpainCalpain in the in the Proteolytic Cleavage of Proteolytic Cleavage of E-cadE-cadherinherin in Prostate and Mamm in Prostate and Mammary Epithelial Cellsary Epithelial Cells

J. Biol. Chem., Vol. 278, Issue 2, 1372-1379, January 10, 2003J. Biol. Chem., Vol. 278, Issue 2, 1372-1379, January 10, 2003 Jonathan Rios-Doria §, Kathleen C. Day ¶, Rainer Kuefer , Michael G. Rashid , Jonathan Rios-Doria §, Kathleen C. Day ¶, Rainer Kuefer , Michael G. Rashid ,

Arul M. Chinnaiyan **¶, Mark A. Rubin **¶, and Mark L. Day §¶Arul M. Chinnaiyan **¶, Mark A. Rubin **¶, and Mark L. Day §¶ From the  Department of Urology and the § Program in Cellular and Molecular From the  Department of Urology and the § Program in Cellular and Molecular

Biology, University of Michigan, the  Department of Urology, University of Ulm, Biology, University of Michigan, the  Department of Urology, University of Ulm, Prittwitz-Strasse 43, 89075 Ulm, Germany, and the ** Department of Pathology, Prittwitz-Strasse 43, 89075 Ulm, Germany, and the ** Department of Pathology, and the ¶ University of Michigan Comprehensive Cancer Center, University of Mand the ¶ University of Michigan Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109 ichigan, Ann Arbor, Michigan 48109

BACKROUNDBACKROUND

E-cadherinE-cadherinThe cadherin structure consists of an extracellular dThe cadherin structure consists of an extracellular domain with five tandem repeats, a transmembrane domain, and omain with five tandem repeats, a transmembrane domain, and a cytoplasmic domain that interacts with the catenin family bindia cytoplasmic domain that interacts with the catenin family binding proteins.ng proteins.

Association of E-cadherin with the catenin family of proteins is cAssociation of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. ritical for the maintenance of a functional adhesive complex.

Inactivation of E-cadherin through gene mutation, transcriptionaInactivation of E-cadherin through gene mutation, transcriptional inactivation, or promoter methylation has been demonstrated il inactivation, or promoter methylation has been demonstrated in many adenocarcinomas n many adenocarcinomas

CalpainCalpain’s structure’s structure The 80 kDa catalytic subunit : The 80 kDa catalytic subunit : dodo

main Imain I is a short pro-domain regi is a short pro-domain region; on; domain IIdomain II represents a distinc represents a distinct papain-like cysteine protease dot papain-like cysteine protease domain; main; domain IIIdomain III has no significan has no significant homology to any other protein, mt homology to any other protein, may regulate protease activity; domay regulate protease activity; domain IV is a calcium binding domain ain IV is a calcium binding domain that has been proposed to play a rthat has been proposed to play a role in substrate recognition. ole in substrate recognition.

The small 30 kDa: The small 30 kDa: domain IVdomain IV of t of the catalytic subunit and he catalytic subunit and domain Vdomain VII of the regulatory subunit, each p of the regulatory subunit, each possess five sets of EF hand calciuossess five sets of EF hand calcium binding motifs that have been pm binding motifs that have been proposed to confer calcium dependroposed to confer calcium dependency upon the activity of calpain. ency upon the activity of calpain.

Calpain is involved in cell-cycle regulatCalpain is involved in cell-cycle regulat

ion, signal transduction, and apoptosis.ion, signal transduction, and apoptosis. Activation of v-Src induces protein syntActivation of v-Src induces protein synt

hesis of calpain leading to calpain-medhesis of calpain leading to calpain-mediated degradation of its own inhibitor ciated degradation of its own inhibitor calpastatin thereby, further enhancing calpastatin thereby, further enhancing calpain activity. alpain activity.

promotes proteolytic cleavage of FAKpromotes proteolytic cleavage of FAK accelerates the progression of transforaccelerates the progression of transfor

med cells through the G1 stage of the med cells through the G1 stage of the cell-cycle and contributes to anchoragcell-cycle and contributes to anchorage-independent growth. e-independent growth.

EGFR induced phosphorylation of calpEGFR induced phosphorylation of calpain and increased calpain activity that ain and increased calpain activity that subsequently increased motility. subsequently increased motility.

Proteins that have been identified as sProteins that have been identified as substrates of calpain include the cytoskubstrates of calpain include the cytoskeletal proteins spectrin and members oeletal proteins spectrin and members of the integrin family.f the integrin family.

E-cadherinE-cadherin 和和 calpaincalpain 均是钙依赖的，也均是钙依赖的，也都与细胞粘附关系密切，但以前的研究并没都与细胞粘附关系密切，但以前的研究并没有将二者联系起来。有将二者联系起来。

文章作者在研究钙离子在上皮钙粘素分解中文章作者在研究钙离子在上皮钙粘素分解中作用的时候，发现了一种新的作用的时候，发现了一种新的 100kda100kda 的钙的钙粘素水解片段，即粘素水解片段，即 E-cad100E-cad100 。。

而而 E-cad100E-cad100 产生可以被产生可以被 calpaincalpain 抑制剂所抑制剂所阻止。所以阻止。所以 E-cadherinE-cadherin 很可能也是很可能也是 calpaicalpainn 的底物。的底物。

PURPOSEPURPOSE

11 、 、 Is E-cadherin a substrate for Is E-cadherin a substrate for calpaincalpain ？？ 22 、 、 Is Is calpaincalpain-dependent proteolysis of -dependent proteolysis of

E-cadherin associated with prostate canceE-cadherin associated with prostate cancer progressionr progression ？？

Cell LinesCell Lines ：： LNCaP , MCF-7, and LNCaP , MCF-7, and SKBR3(no ecad)SKBR3(no ecad) IonomycinIonomycin ：： A divalent calcium ionophore that is widely A divalent calcium ionophore that is widely

used as a tool to investigate the role of intracellular calciuused as a tool to investigate the role of intracellular calcium in cellular processes. m in cellular processes. ↑↑Ca++Ca++

TPATPA ：： 12-o-tetradecanoylphorbol-13-acetate. 12-o-tetradecanoylphorbol-13-acetate. ↑PKC↑PKC AntibodiesAntibodies-- Antibodies used for the detection of E-cadhe-- Antibodies used for the detection of E-cadhe

rin were HECD-1, E-9, 4A2 , C20820, E2, and SC-1499 . rin were HECD-1, E-9, 4A2 , C20820, E2, and SC-1499 . Tissue ProcurementTissue Procurement : fresh radical prostatectomy specim : fresh radical prostatectomy specim

ens were frozen in liquid nitrogen within 30 min after surgiens were frozen in liquid nitrogen within 30 min after surgical excision. A Rapid Autopsy Protocol has been previouslcal excision. A Rapid Autopsy Protocol has been previously described. The metastases were confirmed as prostate cy described. The metastases were confirmed as prostate cancer in origin by a genitourinary pathologist at the Univerancer in origin by a genitourinary pathologist at the University of Michigan.sity of Michigan.

ResultsResults 11 、 、 Ionomycin and Calpain Inhibitor Experiments for Ionomycin and Calpain Inhibitor Experiments for LNCaP , MCF-7 LNCaP , MCF-7 。。

fig1fig1 ：：

ConclusionConclusion ：： Ionomycin Induces a Calpain-dependent ClIonomycin Induces a Calpain-dependent Cleavage of E-cadherin to 100 kDa eavage of E-cadherin to 100 kDa ，， which can be significantlwhich can be significantly inhibited by calpain inhibitors. y inhibited by calpain inhibitors.

22 、 、 purified purified calpaincalpain could generate E-cad100 in vitro .fig2 could generate E-cad100 in vitro .fig2 ：：

ConclusionConclusion ： ： both µ- and m-both µ- and m-calpaincalpain can specifically cleave can specifically cleave mature E-cadherin to the 100-kDa fragment mature E-cadherin to the 100-kDa fragment in vitroin vitro. .

A similar A similar in vitroin vitro assay was performed using purified assay was performed using purified caspase-caspase-33, which failed to demonstrate cleavage of E-cadherin to E-ca, which failed to demonstrate cleavage of E-cadherin to E-cad100 .d100 .

33 、、 Expression and truncation of exogenous E-cadherin in SKBR3 celExpression and truncation of exogenous E-cadherin in SKBR3 cells. ls. AA, Protein extracts from SKBR3 parental cells (, Protein extracts from SKBR3 parental cells (lanes 1-2lanes 1-2) and SK-E) and SK-Ecad cells (cad cells (lanes 3-6lanes 3-6), treated with TPA at the indicated times, were im), treated with TPA at the indicated times, were immunoblotted with the HECD-1 antibody, Fig. 3munoblotted with the HECD-1 antibody, Fig. 3

11 、 、 Activation of PKC in these E-cad-expressing SKBR3 cells (termActivation of PKC in these E-cad-expressing SKBR3 cells (termed SK-Ecad) revealed the rapid accumulation of E-cad100 over a ed SK-Ecad) revealed the rapid accumulation of E-cad100 over a course of 12 h course of 12 h ；；

22 、、 PKC activation by TPA treatment could induce expression of anPKC activation by TPA treatment could induce expression of any endogenous E-cadherin protein in transfected SKBR3 cells.y endogenous E-cadherin protein in transfected SKBR3 cells. the the mechanismmechanism is currently unclearis currently unclear . .

qustionqustion ：： E-cadherin synthesis is required for cleavage E-cadherin synthesis is required for cleavage ？？

44 、、 LNCaP cells were pretreated with LNCaP cells were pretreated with cycloheximidecycloheximide prio prior to TPA treatment r to TPA treatment ：：

Fig. 4.  Fig. 4.   LNCaP cells were pretreated with 50 µM cyclohexi LNCaP cells were pretreated with 50 µM cycloheximide 30 min prior to TPA treatment. 50 µg of protein extracmide 30 min prior to TPA treatment. 50 µg of protein extracts from cells that were harvested at the indicated times werts from cells that were harvested at the indicated times were resolved by 6% SDS-PAGE and immunoblotted with HEe resolved by 6% SDS-PAGE and immunoblotted with HECD-1 antibody. CD-1 antibody. ConclusionConclusion ： ： E-cadherin synthesis is not required foE-cadherin synthesis is not required for cleavage.r cleavage.

55 、、 Epitope and Functional Mapping of E-cad100.Epitope and Functional Mapping of E-cad100.

Fig. 5. Fig. 5. A schematic of full-length E-cadherin and a table of the antibodies and epitopA schematic of full-length E-cadherin and a table of the antibodies and epitopes to which they are mapped are shown. TPA-treated SK-Ecad protein extracts es to which they are mapped are shown. TPA-treated SK-Ecad protein extracts were resolved by SDS-PAGE and immunoblotted with E-9 (site A), 4A2 (site B), were resolved by SDS-PAGE and immunoblotted with E-9 (site A), 4A2 (site B), C20820 (site C), and SC-1499 (site D) antibodies. Lysates were resolved by 6% C20820 (site C), and SC-1499 (site D) antibodies. Lysates were resolved by 6% SDS-PAGESDS-PAGE

ConclusionConclusion ：： the cleavage site was in the cytoplasmic domain of E-cadherthe cleavage site was in the cytoplasmic domain of E-cadherin located in located between the C20820 and SC-1499 epitopesbetween the C20820 and SC-1499 epitopes. .

66 、、 E-cad100 does not associate with the catenin complex.E-cad100 does not associate with the catenin complex. The The ßß -catenin and -catenin and ÝÝ -catenin binding domains : 815-839 of the -catenin binding domains : 815-839 of the cytoplasmic domain. The p120 binding site: 758-773.cytoplasmic domain. The p120 binding site: 758-773.

Fig. 6.TPA-treated LNCaP protein extracts were Fig. 6.TPA-treated LNCaP protein extracts were immunoprecipitated with E-cadherin antibody 4A2, -catenin, immunoprecipitated with E-cadherin antibody 4A2, -catenin, p120, and -catenin antibodies. A control immunoprecipitation p120, and -catenin antibodies. A control immunoprecipitation was also performed using a normal goat IgG antibody and an was also performed using a normal goat IgG antibody and an immunoprecipation using protein A Sepharose beads alone. immunoprecipation using protein A Sepharose beads alone.

ConclusionConclusion :E-cad100 has lost the :E-cad100 has lost the ßß - and - and ÝÝ -catenin binding do -catenin binding domains. mains.

77 、 、 E-cadherin mutagenesis inhibits truncation E-cadherin mutagenesis inhibits truncation in vivoin vivo

Fig. 7.   Fig. 7.   AA, E-cadherin amino acid sequence, from 771-810, is sho, E-cadherin amino acid sequence, from 771-810, is shown with the targeted mutation sites. wn with the targeted mutation sites. BB, the corresponding mutant c, the corresponding mutant constructs containing in-frame internal deletions were stably transfeonstructs containing in-frame internal deletions were stably transfected into SKBR3 cells and protein extracts harvested at the indicatcted into SKBR3 cells and protein extracts harvested at the indicated times following TPA treatment.SKBR3-Ecad wild type cells are ed times following TPA treatment.SKBR3-Ecad wild type cells are also shown (also shown (lane 10lane 10). ). ConclusionConclusion ：： 11 、、 Mutation of the E-cadherin Cytoplasmic DomMutation of the E-cadherin Cytoplasmic Domain Inhibits Truncation of E-cadherin in Vivoain Inhibits Truncation of E-cadherin in Vivo ; ;22 、、 amino acid residamino acid residues 782-787 are essential for ues 782-787 are essential for calpaincalpain cleavage cleavage 。。

88 、 、 m-Calpain is up-regulated in localized and metastatic m-Calpain is up-regulated in localized and metastatic prostate tumors.prostate tumors.

Fig. 8.  Fig. 8.   cDNA microarray analysis was performed using 26 beni cDNA microarray analysis was performed using 26 benign prostate, 49 localized prostate cancer, and 19 metastatic prosgn prostate, 49 localized prostate cancer, and 19 metastatic prostate cancer samples. m-tate cancer samples. m-CalpainCalpain transcript levels were found to b transcript levels were found to be significantly up-regulated in localized prostate and metastatic ce significantly up-regulated in localized prostate and metastatic cancer samples compared with benign prostate. ancer samples compared with benign prostate.

99 、、 E-cad100 is detected in localized and metastatic prostate cancerE-cad100 is detected in localized and metastatic prostate cancer ：：

Fig 9. AFig 9. A, protein extracts (30 µg) of prostate tissue from two different patie, protein extracts (30 µg) of prostate tissue from two different patients were analyzed for E-cadherin expression. Extracts from both the normnts were analyzed for E-cadherin expression. Extracts from both the normal and tumor aspect of the prostate gland were run on a 6% SDS-PAGE geal and tumor aspect of the prostate gland were run on a 6% SDS-PAGE gel and immunoblotted with HECD-1 antibody. l and immunoblotted with HECD-1 antibody. BB, protein extracts were prepa, protein extracts were prepared from metastatic lesions removed from the following sites: red from metastatic lesions removed from the following sites: 11, liver; , liver; 22, dia, diaphragm; phragm; 33, soft tissue; , soft tissue; 44, peritoneum; , peritoneum; 55, adrenal gland; , adrenal gland; 66, paratracheal lym, paratracheal lymph node .ph node .

ConclusionConclusion : :increased expression of increased expression of calpaincalpain is associated with the ge is associated with the generation of the 100-kDa fragment in localized and metastatic disease. neration of the 100-kDa fragment in localized and metastatic disease.

DiscussionDiscussion

CalpainCalpain induces a specific, inactivating proteolytic c induces a specific, inactivating proteolytic cleavage within the cytoplasmic domain of E-cadherileavage within the cytoplasmic domain of E-cadherin in prostate and mammary epithelial cells. Pretreatn in prostate and mammary epithelial cells. Pretreatment of LNCaP and MCF-7 cells with ment of LNCaP and MCF-7 cells with calpaincalpain inhibit inhibitors blocked the generation of ionomycin-induced E-ors blocked the generation of ionomycin-induced E-cad100, strongly implicating cad100, strongly implicating calpaincalpain in this process. in this process.

In vitroIn vitro experiments also supported the hypothesis t experiments also supported the hypothesis that E-cadherin is a substrate for hat E-cadherin is a substrate for calpaincalpain. Both µ- a. Both µ- and m-nd m-calpaincalpain effectively cleaved full-length E-cadhe effectively cleaved full-length E-cadherin rin in vitroin vitro to 100 kDa. to 100 kDa.

This truncation occurred in the cytoplasmic domain This truncation occurred in the cytoplasmic domain of E-cadherin, and that this truncated species of E-cof E-cadherin, and that this truncated species of E-cadherin is unable to bind to adherin is unable to bind to ßß -catenin, -catenin, ÝÝ -catenin, a -catenin, and p120, which are essential for the adhesive and cnd p120, which are essential for the adhesive and cell signaling functions of E-cadherin. ell signaling functions of E-cadherin.

The residues 782-787 are either critical for The residues 782-787 are either critical for calpaincalpain binding or represent the actual cleavage site.binding or represent the actual cleavage site.

Based on these observations, we hypothesized that Based on these observations, we hypothesized that

specific proteolytic cleavage events, such as that mspecific proteolytic cleavage events, such as that mediated by calpain, target and inactivate E-cadherin.ediated by calpain, target and inactivate E-cadherin. Such a loss of E-cadherin function may result in th Such a loss of E-cadherin function may result in the net reduction of interepithelial adhesion and prome net reduction of interepithelial adhesion and promote the malignant transformation of prostate epitheliote the malignant transformation of prostate epithelial cells.al cells.

Over.Over.

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