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Differential subcellular localization renders HAI-2 a matriptase inhibitor in breast cancer cells but not in mammary epithelial cells.

Chang HH, Xu Y, Lai H, Yang X, Tseng CC, Lai YJ, Pan Y, Zhou E, Johnson MD, Wang JK, Lin CY - PLoS ONE (2015)

Bottom Line: The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1.Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts.HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1.

View Article: PubMed Central - PubMed

Affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology Georgetown University Washington, DC, 20057, United States of America; Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, ROC.

ABSTRACT
The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.

No MeSH data available.


Related in: MedlinePlus

In Solution, HAI-2 is a better matriptase inhibitor than HAI-1.A. Active matriptase prepared from Ramos human B-cell lymphoma cells was mixed with 184 A1N4 human mammary epithelial cell lysate and incubated at 37°C for 10 min. Active matriptase preparation alone, cell lysate alone, and the mixture were analyzed for matriptase tryptic activity by cleavage of the fluorogenic substrate Boc-Gln-Ala-Arg-AMC. RFU stands for relative fluorescent units. B. The active matriptase preparation was mixed with matriptase-depleted 184 A1N4 cell lysates and at 37°C for 10 min to allow inhibition of matriptase by the HAIs. The mixture was divided and subjected to immunodepletion of HAI-1 species using the HAI-1 mAb M19 or HAI-2 species using the HAI-2 mAb DC16. The active matriptase preparation (lanes 1), the matriptase-depleted 184 A1N4 cell lysate (lanes 2), the mixture (lanes 3), the HAI-1 depleted mixture, and the HAI-2 depleted mixture were analyzed for matriptase species, HAI-1 species, and HAI-2 species by immunoblot using the matriptase mAb M24 (Total MTP), the HAI-1 mAb M19 (HAI-1) and the HAI-2 mAb DC16 (HAI-2), respectively.
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pone.0120489.g002: In Solution, HAI-2 is a better matriptase inhibitor than HAI-1.A. Active matriptase prepared from Ramos human B-cell lymphoma cells was mixed with 184 A1N4 human mammary epithelial cell lysate and incubated at 37°C for 10 min. Active matriptase preparation alone, cell lysate alone, and the mixture were analyzed for matriptase tryptic activity by cleavage of the fluorogenic substrate Boc-Gln-Ala-Arg-AMC. RFU stands for relative fluorescent units. B. The active matriptase preparation was mixed with matriptase-depleted 184 A1N4 cell lysates and at 37°C for 10 min to allow inhibition of matriptase by the HAIs. The mixture was divided and subjected to immunodepletion of HAI-1 species using the HAI-1 mAb M19 or HAI-2 species using the HAI-2 mAb DC16. The active matriptase preparation (lanes 1), the matriptase-depleted 184 A1N4 cell lysate (lanes 2), the mixture (lanes 3), the HAI-1 depleted mixture, and the HAI-2 depleted mixture were analyzed for matriptase species, HAI-1 species, and HAI-2 species by immunoblot using the matriptase mAb M24 (Total MTP), the HAI-1 mAb M19 (HAI-1) and the HAI-2 mAb DC16 (HAI-2), respectively.

Mentions: Next, we compared the ability of cell-derived HAI-1 and HAI-2 to inhibit active matriptase. Free active matriptase along with matriptase zymogen was prepared from the material shed from Ramos human Burkitt lymphoma cells exposed to a pH 6.0 buffer, in order to induce matriptase activation. Active matriptase represents the vast majority of the tryptic activity in this shed fraction, which can be detected by cleavage of the synthetic fluorogenic substrate Boc-Gln-Ala-Arg-AMC (Fig. 2A, Blue Diamond), and which can be immunodepleted by a matriptase mAb, as demonstrated previously [28]. Both HAI-1 and HAI-2 were prepared from 184 A1N4 mammary epithelial cell lysates, from which the endogenous matriptase had been removed by immunodepletion using matriptase mAb 21–9 conjugated to Sepharose beads (Fig. 2B, lanes 2) and contained no tryptic activity capable of cleaving Boc-Gln-Ala-Arg-AMC (Fig. 2A Red Rectangle). Incubation of the 184 A1N4 preparation with the free active matriptase prepared from Ramos cells at 37°C for 10 minutes, resulted in a sample in which matriptase tryptic activity was significant inhibited (Fig. 2A, Green Triangle). Analysis of this sample by immunoblot assay demonstrated that the formation of matriptase complexes of around 100- and 120-kDa (Fig. 2B, Total MTP, lane 3) was associated with the suppression of tryptic activity. Using the HAI-1 mAb M19 and HAI-2 mAb DC16, we also showed that a HAI-1 complex of 120-kDa (Fig. 2B HAI-1, lane 3) and two HAI-2 complexes of 100- and 130-kDa had been formed (Fig. 2B HAI-2, lane 3). The 120-kDa matriptase-HAI-1 complex and the 130-kDa matriptase-HAI-2 complex appear as one large merged band in the immunoblot analysis using the matriptase mAb (Fig. 2B, Total MTP, lane 3). To further confirm the identity of these three HAI-matriptase complexes present in the sample (Fig. 2B, lanes 3) we either depleted the HAI-1 species from the sample with the HAI-1 mAb (Fig. 2B, HAI-1, lane 4) or depleted the HAI-2 species with the HAI-2 mAb (Fig. 2B, HAI-2, lane 5). Along with the depletion of HAI-1 species (Fig. 2B, HAI-1 lane 4), the lower portion of the 120-kDa matriptase complex was depleted (Fig. 2B, total MTP, lane 4). The specificity of the HAI-1 immunodepletion was confirmed by the continued presence of the 70-kDa matriptase zymogen and the two matriptase-HAI-2 complexes (Fig. 2B, total MTP, lane 4; HAI-2, lane 4). These analyses confirm that the lower portion of the 120-kDa matriptase containing complex is matriptase bound with HAI-1 and its presence indicates that matriptase is being inhibited by interacting with HAI-1. Similarly, concurrent with the immunodepletion of HAI-2 containing species (Fig. 2B, HAI-2, lane 5), the upper portion of the 120-kDa and the 100-kDa species detected with the matriptase antibody were removed (Fig. 2B, total MTP, lane 5), suggesting that these two matriptase complexes contain HAI-2. Remaining after HAI-2 immunodepletion was the 70-kDa matriptase zymogen, free HAI-1 and the 120-kDa matriptase-HAI-1 complex (Fig. 2B, lanes 5 under total MTP and HAI-1,) which again verifies the specificity of immunodepletion. These data suggest that the formation of the two matriptase-HAI-2 complexes contributes to the inhibition of active matriptase. These data also imply that HAI-2 may be a more potent inhibitor than HAI-1 in solution. We base this supposition on the relative ratio of the abundance of the matriptase-HAI-1 complex versus matriptase-HAI-2 complexes compared to the ratio of the uncomplexed HAI-1 versus HAI-2. The matriptase-HAI-1:matriptase-HAI-2 ratio was determined to be around 1:2.5 by densitometry and analysis using ImageJ of the bands representing the 120-kDa (Fig. 2B, MTP, lane 5), and the 100-, and 130-kDa (Fig. 2B, total MTP, lane 4) matriptase complexes followed by normalization of these values by the value for the 70-kDa matriptase zymogen bands (Fig. 2B, MTP, lanes 4 and 5). In addition we observed that there is very little free HAI-2, the majority being bound to active matriptase to form the two complexes (Fig. 2B, HAI-2 lane 3), whereas only 18% of the available HAI-1 was bound to matriptase in the 120-kDa complex (Fig. 2B, HAI-1, lane 3). Thus, in 184 A1N4 cells the amount of HAI-1 is about 2.2 (1/2.5÷0.18 = 2.2) times greater than HAI-2 prior to the incubation with active matriptase, which is in marked contrast to the matriptase-HAI-1:matriptase-HAI-2 ratio (1:2.5) after incubation with active matriptase. Collectively, these data suggest that both HAI-1 and HAI-2 are capable forming stable complexes with active matriptase in solution and that cellular HAI-2 appears to be a more effective matriptase inhibitor than cellular HAI-1. In addition, given the molecular masses of the proteins, the stoichiometry of the matriptase complexes appears to be 1:1 for the 100-kDa matriptase-HAI-2 complex and the 120-kDa matriptase-HAI-1complex. It seems likely that there must be a third protein in addition to matriptase in the 130-kDa HAI-2 complex. We summarize the experiment in Fig. 2C.


Differential subcellular localization renders HAI-2 a matriptase inhibitor in breast cancer cells but not in mammary epithelial cells.

Chang HH, Xu Y, Lai H, Yang X, Tseng CC, Lai YJ, Pan Y, Zhou E, Johnson MD, Wang JK, Lin CY - PLoS ONE (2015)

In Solution, HAI-2 is a better matriptase inhibitor than HAI-1.A. Active matriptase prepared from Ramos human B-cell lymphoma cells was mixed with 184 A1N4 human mammary epithelial cell lysate and incubated at 37°C for 10 min. Active matriptase preparation alone, cell lysate alone, and the mixture were analyzed for matriptase tryptic activity by cleavage of the fluorogenic substrate Boc-Gln-Ala-Arg-AMC. RFU stands for relative fluorescent units. B. The active matriptase preparation was mixed with matriptase-depleted 184 A1N4 cell lysates and at 37°C for 10 min to allow inhibition of matriptase by the HAIs. The mixture was divided and subjected to immunodepletion of HAI-1 species using the HAI-1 mAb M19 or HAI-2 species using the HAI-2 mAb DC16. The active matriptase preparation (lanes 1), the matriptase-depleted 184 A1N4 cell lysate (lanes 2), the mixture (lanes 3), the HAI-1 depleted mixture, and the HAI-2 depleted mixture were analyzed for matriptase species, HAI-1 species, and HAI-2 species by immunoblot using the matriptase mAb M24 (Total MTP), the HAI-1 mAb M19 (HAI-1) and the HAI-2 mAb DC16 (HAI-2), respectively.
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Related In: Results  -  Collection

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pone.0120489.g002: In Solution, HAI-2 is a better matriptase inhibitor than HAI-1.A. Active matriptase prepared from Ramos human B-cell lymphoma cells was mixed with 184 A1N4 human mammary epithelial cell lysate and incubated at 37°C for 10 min. Active matriptase preparation alone, cell lysate alone, and the mixture were analyzed for matriptase tryptic activity by cleavage of the fluorogenic substrate Boc-Gln-Ala-Arg-AMC. RFU stands for relative fluorescent units. B. The active matriptase preparation was mixed with matriptase-depleted 184 A1N4 cell lysates and at 37°C for 10 min to allow inhibition of matriptase by the HAIs. The mixture was divided and subjected to immunodepletion of HAI-1 species using the HAI-1 mAb M19 or HAI-2 species using the HAI-2 mAb DC16. The active matriptase preparation (lanes 1), the matriptase-depleted 184 A1N4 cell lysate (lanes 2), the mixture (lanes 3), the HAI-1 depleted mixture, and the HAI-2 depleted mixture were analyzed for matriptase species, HAI-1 species, and HAI-2 species by immunoblot using the matriptase mAb M24 (Total MTP), the HAI-1 mAb M19 (HAI-1) and the HAI-2 mAb DC16 (HAI-2), respectively.
Mentions: Next, we compared the ability of cell-derived HAI-1 and HAI-2 to inhibit active matriptase. Free active matriptase along with matriptase zymogen was prepared from the material shed from Ramos human Burkitt lymphoma cells exposed to a pH 6.0 buffer, in order to induce matriptase activation. Active matriptase represents the vast majority of the tryptic activity in this shed fraction, which can be detected by cleavage of the synthetic fluorogenic substrate Boc-Gln-Ala-Arg-AMC (Fig. 2A, Blue Diamond), and which can be immunodepleted by a matriptase mAb, as demonstrated previously [28]. Both HAI-1 and HAI-2 were prepared from 184 A1N4 mammary epithelial cell lysates, from which the endogenous matriptase had been removed by immunodepletion using matriptase mAb 21–9 conjugated to Sepharose beads (Fig. 2B, lanes 2) and contained no tryptic activity capable of cleaving Boc-Gln-Ala-Arg-AMC (Fig. 2A Red Rectangle). Incubation of the 184 A1N4 preparation with the free active matriptase prepared from Ramos cells at 37°C for 10 minutes, resulted in a sample in which matriptase tryptic activity was significant inhibited (Fig. 2A, Green Triangle). Analysis of this sample by immunoblot assay demonstrated that the formation of matriptase complexes of around 100- and 120-kDa (Fig. 2B, Total MTP, lane 3) was associated with the suppression of tryptic activity. Using the HAI-1 mAb M19 and HAI-2 mAb DC16, we also showed that a HAI-1 complex of 120-kDa (Fig. 2B HAI-1, lane 3) and two HAI-2 complexes of 100- and 130-kDa had been formed (Fig. 2B HAI-2, lane 3). The 120-kDa matriptase-HAI-1 complex and the 130-kDa matriptase-HAI-2 complex appear as one large merged band in the immunoblot analysis using the matriptase mAb (Fig. 2B, Total MTP, lane 3). To further confirm the identity of these three HAI-matriptase complexes present in the sample (Fig. 2B, lanes 3) we either depleted the HAI-1 species from the sample with the HAI-1 mAb (Fig. 2B, HAI-1, lane 4) or depleted the HAI-2 species with the HAI-2 mAb (Fig. 2B, HAI-2, lane 5). Along with the depletion of HAI-1 species (Fig. 2B, HAI-1 lane 4), the lower portion of the 120-kDa matriptase complex was depleted (Fig. 2B, total MTP, lane 4). The specificity of the HAI-1 immunodepletion was confirmed by the continued presence of the 70-kDa matriptase zymogen and the two matriptase-HAI-2 complexes (Fig. 2B, total MTP, lane 4; HAI-2, lane 4). These analyses confirm that the lower portion of the 120-kDa matriptase containing complex is matriptase bound with HAI-1 and its presence indicates that matriptase is being inhibited by interacting with HAI-1. Similarly, concurrent with the immunodepletion of HAI-2 containing species (Fig. 2B, HAI-2, lane 5), the upper portion of the 120-kDa and the 100-kDa species detected with the matriptase antibody were removed (Fig. 2B, total MTP, lane 5), suggesting that these two matriptase complexes contain HAI-2. Remaining after HAI-2 immunodepletion was the 70-kDa matriptase zymogen, free HAI-1 and the 120-kDa matriptase-HAI-1 complex (Fig. 2B, lanes 5 under total MTP and HAI-1,) which again verifies the specificity of immunodepletion. These data suggest that the formation of the two matriptase-HAI-2 complexes contributes to the inhibition of active matriptase. These data also imply that HAI-2 may be a more potent inhibitor than HAI-1 in solution. We base this supposition on the relative ratio of the abundance of the matriptase-HAI-1 complex versus matriptase-HAI-2 complexes compared to the ratio of the uncomplexed HAI-1 versus HAI-2. The matriptase-HAI-1:matriptase-HAI-2 ratio was determined to be around 1:2.5 by densitometry and analysis using ImageJ of the bands representing the 120-kDa (Fig. 2B, MTP, lane 5), and the 100-, and 130-kDa (Fig. 2B, total MTP, lane 4) matriptase complexes followed by normalization of these values by the value for the 70-kDa matriptase zymogen bands (Fig. 2B, MTP, lanes 4 and 5). In addition we observed that there is very little free HAI-2, the majority being bound to active matriptase to form the two complexes (Fig. 2B, HAI-2 lane 3), whereas only 18% of the available HAI-1 was bound to matriptase in the 120-kDa complex (Fig. 2B, HAI-1, lane 3). Thus, in 184 A1N4 cells the amount of HAI-1 is about 2.2 (1/2.5÷0.18 = 2.2) times greater than HAI-2 prior to the incubation with active matriptase, which is in marked contrast to the matriptase-HAI-1:matriptase-HAI-2 ratio (1:2.5) after incubation with active matriptase. Collectively, these data suggest that both HAI-1 and HAI-2 are capable forming stable complexes with active matriptase in solution and that cellular HAI-2 appears to be a more effective matriptase inhibitor than cellular HAI-1. In addition, given the molecular masses of the proteins, the stoichiometry of the matriptase complexes appears to be 1:1 for the 100-kDa matriptase-HAI-2 complex and the 120-kDa matriptase-HAI-1complex. It seems likely that there must be a third protein in addition to matriptase in the 130-kDa HAI-2 complex. We summarize the experiment in Fig. 2C.

Bottom Line: The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1.Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts.HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1.

View Article: PubMed Central - PubMed

Affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology Georgetown University Washington, DC, 20057, United States of America; Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, ROC.

ABSTRACT
The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.

No MeSH data available.


Related in: MedlinePlus