Elucidating the function of secreted maspin: inhibiting cathepsin D-mediated matrix degradation

Abstract

Cellular interaction with the extracellular milieu plays a significant role in normal biological and pathologic processes. Excessive degradation of basement membrane matrix by proteolytic enzymes is a hallmark of tumor invasion and metastasis, and aspartyl proteinase cathepsin D is implicated as a major contributor to this process. Maspin, a non-inhibitory serpin, plays an important role in mammary gland development and remodeling. Expression of Maspin is decreased in primary tumors and lost in metastatic lesions. Maspin is mostly cytoplasmic and is partially secreted; however, the fate and function of secreted Maspin has remained mostly unexplored. We hypothesized that secreted Maspin is incorporated into the matrix deposited by normal mammary epithelial cells and thus could play a critical role in cathepsin D-mediated matrix degradation and remodeling of mammary tissue. In the absence of Maspin, as is the case with most cancer cells, matrix degradation proceeds unrestricted, thus facilitating the progression to metastasis. To test this, we employed an in vitro model where gels containing both types I and IV collagen were preconditioned with normal mammary epithelial cells to allow the incorporation of secreted Maspin. This conditioned matrix was used to examine cathepsin D-mediated collagen degradation by human breast cancer cell lines. Our results indicate that secretion of Maspin and its deposition into the extracellular milieu play an important role in matrix degradation. In this capacity, Maspin could potentially regulate mammary tissue remodeling occurring under normal and pathologic conditions. In addition, these findings could have a potential effect on future therapeutic intervention strategies for breast cancer.

Figure 1

A, Western blot analysis to detect Maspin in the conditioned medium (CM) and the matrix deposited (Deposited matrix) by normal mammary epithelial cells (HMEC 1330) and its incorporation into a three-dimensional collagen (Col) matrix (HMEC-conditioned matrix). r-Maspin at concentrations indicated was used to examine the level of Maspin secreted into the conditioned medium and/or deposited in the matrix. B, the presence of Maspin in the HMEC-conditioned matrix was further confirmed by immunohistochemical analysis of the matrix following the complete removal of the cells. Breast cancer cell lines MCF-7 and MDA-MB-231 were plated on collagen I matrix in 96-well culture dishes (75 × 10³ per well) for 4 d. C, the conditioned medium was collected and concentrated 10× (MCF-7) or 2× (MDA-MB-231) and subjected to Western blot analysis to detect secreted pro-cathepsin D (pro-CathD) and Maspin. D, the cells were then removed by treatment with 20 mmol/L NH₄OH; the remaining collagen matrices were washed with PBS and boiled in SDS-PAGE sample buffer and subjected to Western blot analysis for the detection of cathepsin D. The cathepsin D detected in the conditioned medium is the pro-form (~52–54 kDa), whereas that in the matrix is the intermediary and mature active forms.

Figure 2

A, r-Maspin was incorporated into collagen solution before the gelation process at concentrations ranging from 100 to 500 ng per well in 96-well culture dishes. MDA-MB-231 cells were plated on these gels as described in Fig. 1 legend. Analysis of the matrix by Western blot for the presence of Maspin and cathepsin D (and actin as loading control) is depicted in Fig. 1A. For comparison purposes, bovine serum albumin (BSA)–incorporated (500 ng per well) collagen matrix is also included and indicates minimal changes in cathepsin D levels. B, for immunohistochemical analysis, collagen matrices were cast in an eight-well culture dish with or without r-Maspin (500 ng per well). MDA-MB-231 cells were plated on these matrices at 10⁵ per well for a period of 4 d. The cells were then removed by NH₄OH treatment (as described in the Materials and Methods), and the remaining matrices were fixed and treated with antibodies to cathepsin D. C, culturing MDA-MB-231 breast cancer cells on Maspin-incorporated collagen matrix is associated with a reduction in cathepsin D mRNA within 24 h, which returns to baseline by 48 h. D, for quantitative estimation of collagen matrix degradation, fluorescein-conjugated collagen I (DQ collagen) was incorporated into the matrix, and the fluorescent fragments released from the proteolytic cleavage of collagen I were quantified by an ELISA reader. For comparison, collagen matrix degradation resulting from Maspin-transfected MCF-7 or MDA-MB-231 are included. Inset, Western blot analysis of post-nuclear fraction of Maspin-transfected MCF-7 and MDA-MB-231 cells to show Maspin expression.

Figure 3

Three different recombinant mutated Maspin(s) were incorporated into collagen matrix as described in Fig. 2 legend. MDA-MB-231 cells were plated on these matrices in 96-well culture dishes (75 × 10³ per well) for 4 d. A, the conditioned medium was collected, concentrated 2×, and subjected to Western blot analysis to detect secreted pro-cathepsin D and r-mutated Maspin. B, the cells were removed by treatment with 20 mmol/L NH₄OH, and the denuded matrices were washed with PBS and boiled in SDS-PAGE sample buffer and subjected to Western blot analysis for the detection of matrix-associated cathepsin D and r-mutated Maspin. Actin was used as loading control. rR/A-Maspin, recombinant Maspin mutated at Arg³⁴¹ to alanine; rK/H-Maspin, recombinant Maspin mutated at Lys³⁴⁶ to histidine; rY/F-Maspin, recombinant Maspin mutated at Tyr³⁵⁷ to phenylalanine (generated in our laboratory). The position of 45- and 37-kDa molecular weight markers are indicated.