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1Institute of Anatomy and 4Institute of Biochemistry, University of Leipzig, Leipzig, Germany; 2Laboratory of Molecular Biology and Genetic Engineering, University of Liege, Liege, Belgium; and 3Department of Medical Sciences, University "Amedeo Avogadro", Novara, Italy
Submitted 3 May 2007 ; accepted in final form 14 August 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FRLK(Dnp)-D-R-NH2 was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis. prolactin fragments; lysosomal proteases; acidic microenvironment; extracellular proteolysis; ovary; cow
Lysosomal Cath D is synthesized as an inactive precursor, procathepsin D, in the rough endoplasmic reticulum. It is translocated to the Golgi apparatus and finally targeted to the lysosomes, thanks to the presence of acquired mannose-6-phosphate residues (32). There, procathepsin D is converted to an active 44,000 (44K) to 47,000 (47K) single-chain form, which, depending on the species, is further cleaved into an active two-chain form (27). Procathepsin D can escape transfer to lysosomes and can be secreted instead into the extracellular space. Secreted procathepsin D is detected at higher levels in supernatants of tumor cell cultures than in supernatants of normal cell cultures (55). In malignant tumors, the presence of active Cath D forms in the extracellular space is well documented (42, 90). Furthermore, physiological release of active Cath D forms has recently been described in enriched mammary epithelial cell preparations (42, 50) and in various tissues (65). Proton extruders can trigger PRL cleavage under certain physiological conditions (65). Extracellular Cath D might thus participate in generating bioactive proteins under such conditions. In addition, it might be involved in cell migration and proliferation through actions other than proteolytic cleavage (34, 48, 90).
The corpus luteum (CL) is one of the few organs in the adult body that exhibits angiogenesis and angioregression under physiological conditions. Capillary formation and maintenance occur during formation and functioning of the cyclic CL and the CL of pregnancy (19). Along with other autocrine-paracrine angiogenic factors (e.g., prostaglandin E2, transforming growth factor-
1, basic fibroblast growth factor, vascular endothelial growth factor, and angiogenin), the 23K PRL may contribute to establishing the microvascular bed. This was supported by the finding of Gaytan et al. (31) that PRL treatment can enhance the proliferative activity of CL endothelial cells (EC) and even more by the demonstration by Grosdemouge et al. (37) that angiogenesis during CL development is strongly inhibited in mice with targeted disruption of the PRL-receptor gene.
Blood vessel breakdown and EC loss are inherent features of CL regression (53). PGF2
of uterine origin is the key luteolytic hormone in hoofed ruminants such as cows (53, 57). It initiates a rapid decline in luteal progesterone (functional luteolysis), followed by structural CL regression (structural luteolysis). It acts in concert with other vasoconstrictors such as endothelin-1 and ANG II (75), causing the levels of these factors to rise in the CL (33). Long-term vasoconstriction of luteal arterioles is followed by endothelial injury, EC depletion, blood vessel occlusion, degenerative changes (54, 59), and influx of cytokine- and protease-releasing leukocytes, notably macrophages (38, 54). The observed changes resemble hypoxic damage in other tissues, in which lysosomes participate (20, 21, 38, 93). Not surprisingly, therefore, in various species (47, 67) including ruminants (23, 81), cells of the regressing CL show an increased lysosome content, increased lysosomal enzyme activity, and a release of enzymes from fragile lysosomes into the cytosol of degenerating luteal cells. In a hypoxic microenvironment, full-length 23K PRL is likely to be cleaved into 14K and 16K PRL and thus to participate in the involution process.
In the present study, we have examined whether PRL is synthesized by bovine CL-derived EC and granulosa-like cells (GLC), whether it undergoes posttranslational processing by Cath D, and how full-length PRL and its fragments affect CL-derived EC in vitro. Both of the studied cell types have been extensively characterized by our group (78–80). We show that PRL is synthesized in the bovine CL and that full-length PRL declines during the estrous cycle concomitantly with an increase in total active Cath D. We demonstrate that the CL is equipped enzymatically to generate 16K PRL fragments and that CL tissue extracts do generate PRL fragments similar to those produced by bovine Cath D. Our data suggest that PRL cleavage may contribute to the CL regression mechanism.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The collected CL were assigned to early- (days 1–4 after ovulation), mid- (days 5–17), and late- (days 18–21) luteal stages of the estrous cycle according to their macroscopic appearance (41). These assignments were confirmed by histological examination (41, 69).
CL pieces to be used in molecular biology experiments were immediately shock frozen in liquid nitrogen. CL for cell isolation were transported in ice-cold PBS (pH 7.4) to the laboratory, where they were dissected and mechanically disrupted as described in detail (78–80). Briefly, CL were chopped up into small pieces, first with a scalpel and then with a multiblade knife. The material was suspended in medium, further broken up by vigorous pipetting, and filtered through a 150-µm-pore-size metal sieve and then a 70-µm-pore-size nylon sieve. Cells having passed through these filters were pelleted and resuspended in DMEM mixed 1:1 with Ham's F-12 medium (Invitrogen-GIBCO Cell Culture Products, Karlsruhe, Germany), adjusted to pH 7.2 with 15 mM HEPES and 22 mM NaHCO3 supplemented with 5% FCS. Cells were seeded under semiclonal conditions onto 24-well culture plates precoated with collagen I (1% Vitrogen; Nutacon, Leimuiden, The Netherlands). Nonadherent cells and erythrocytes were removed by medium changes at 24 h and every 4 days thereafter. Between weeks 1 and 4, colonies of CL-derived EC and GLC were obtained, trypsinized, dislodged, and transferred to new culture dishes (1:4 split ratio). Pure cultures showing the marker characteristics of either endothelial or steroidogenic cells (see Fig. 1) were harvested at confluency. The cells were stored in liquid nitrogen until use. At this time, they were replated in either 24- or 96-well culture plates or in 75-cm2 flasks.
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Detection of PRL Transcripts
PRL primer. Primer pairs were chosen from different exons to avoid misinterpretation of results due to possible contaminating genomic DNA. The upstream sense primer (5'-GGGCAGTCATGGTGTCCCACTA-3') is located in exon 2 (GenBank accession no. AY337764) and the downstream antisense primer (5'-AAGTGTCAATCTTGCTTGAATC-3') in exon 5 (GenBank accession no. M34535) of the bovine PRL gene (15).
RT-PCR. Total RNA was reverse transcribed and probed with the primer pair. Briefly, total RNA was prepared from shock-frozen CL tissue and cultured cells by means of the pegGold RNAPure kit (PEQLAB Bitotechnology, Erlangen, Germany). This was followed by chloroform extraction and alcohol precipitation. Reverse transcription was carried out in a final volume of 20 µl containing 5 µg DNase I-treated (2 U/5 µg RNA) RNA, 500 ng oligo(dT)15 primers (Promega, Mannheim, Germany), 4 µl first-strand buffer (Invitrogen), 2 µl of 0.1 M DTT, 1 µl of dNTP mix (10 mM each), 1 µl of RNaseOut (Invitrogen), and 200 U of SuperScript reverse transcriptase (Invitrogen). PCRs were performed in a reaction mixture (25 µl final volume) containing 1 µl of generated cDNA, 0.5 µM sense and antisense primers, 2.5 µl of PCR buffer (10x; Roche, Mannheim, Germany), 2 µl dNTP mix, and 2.5 U Taq DNA polymerase (Roche). Initial denaturing at 94°C for 4 min was followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. PCR products were run on 2% agarose gels stained with ethidium bromide and visualized under UV light. As an internal control, the housekeeping gene GAPDH was amplified in a separate reaction using the same cDNA and 25 cycles. The corresponding upstream and downstream primers were based on the bovine GAPDH sequence (GenBank accession no. NM_001034034) and were 5'-TGAAGGGTGGCGCCAAGAGG-3' and 5'-TGCCAGCCCCAGCATCGAAG-3', respectively. Amplified PCR products were cloned into pGEM-T (Promega). Transformants harboring inserts of adequate size were identified and analyzed by DNA sequencing and interrogation of GenBank.
Detection of PRL and Cath D Proteins and Fragments
Preparation of total tissue protein extracts. Shock-frozen tissue pieces were ground with a mortar and pestle and immediately suspended in lysis buffer (10 mM Tris·HCl, 1 mM EDTA, 1 mM MgCl2, 0.02% Triton X-100) containing freshly added protease inhibitors (protease inhibitor cocktail and 1% PMSF, both from Sigma-Aldrich). The suspension was homogenized with the Ultra Turrax T8 homogenizer and centrifuged in a table centrifuge (16,000 g, 4°C, 15 min). The protein concentration of the supernatant was determined with the Micro BCA protein assay kit (Perbio Science, Bonn, Germany).
Preparation of cultured cell protein extracts. Cells were cultured to confluency in 75-cm2 flasks. Culture medium was removed, and adherent cells were washed with PBS, scraped off, and transferred to supplemented lysis buffer. The crude cell lysate was homogenized and centrifuged; the protein concentration was determined in the supernatant.
Preparation of culture supernatant concentrates. Cells were grown to confluency in 24-well culture plates. After a 24-h incubation in serum-free culture medium, the supernatants of four wells (2 ml) were collected, concentrated in Centricon-YM 10 filter devices (Millipore, Schwalbach, Germany) at 8,500 g, and adjusted to a final volume of 150 µl.
Antibodies. For each protein of interest, several antibodies were used to investigate protein synthesis and processing. Three different anti-bovine PRL MAbs recognizing epitopes at the NH2- (5G2 and 4C10) or COOH-terminal end (6F11) of PRL (73) were used to distinguish NH2-terminal from COOH-terminal fragments after digestion of the full-length protein. MAbs were obtained from Acris Antibodies (Hiddenausen, Germany) or kindly donated by Dr. Jonathan G. Scammell (Department of Pharmacology, University of South Alabama). In addition, we used a polyclonal anti-bovine PRL antiserum. This antiserum is part of the kit distributed by the National Hormone and Peptide Program (NHPP; Dr. A. F. Parlow, Harbor-UCLA Medical Center, Torrance, CA) for RIA determinations of bovine PRL. The ability of the antiserum to react with NH2-terminal bovine PRL fragments of various size is documented in the literature (15). The anti-bovine PRL MAbs were used at a final concentration of 0.2 µg/ml and the anti-bovine PRL antiserum at a final dilution of 1:3,000 for Western blotting.
A rabbit anti-Cath D polyclonal antibody produced in the laboratory of Isidoro and colleagues (12), the rabbit anti-human Cath D antiserum Ab-2 obtained from Merck Biosciences (Bad Soden, Germany), and a rabbit anti-bovine Cath D antiserum (51) kindly donated by Dr. Hoflack (Biotechnological Centre, Dresden University of Technology, Dresden, Germany) were used to detect inactive procathepsin D and its cleaved fragments. The antisera were used at a final concentration of 0.1 µg/ml (Ab-2) or at a final dilution of 1:1,000.
Protein electrophoreses and blotting. Protein samples were boiled in denaturing loading buffer at 95°C for 5 min. Either 20 µg of total protein lysate or 20 µl of concentrated supernatant were loaded per lane of a 15% SDS-PAGE minigel and separated in parallel with a molecular weight marker (Rainbow molecular weight marker; Amersham Bioscience, Freiburg, Germany). Electrophoresed proteins were semi-dry blotted onto a 0.45-µm-pore-size nitrocellulose membrane (Biometra, Göttingen, Germany). Protein loading and transfer efficiency were checked by Ponceau red staining. Destained membranes were blocked with 5% (wt/vol) nonfat dried milk powder at room temperature for 1 h. Subsequently, the protein of interest was labeled overnight at 4°C with the specific primary antibody or antiserum. This was followed by incubation for 1 h at room temperature with the relevant peroxidase-conjugated secondary antibody (0.2 µg/ml). Binding sites were visualized on Hyperfilm ECL (Amersham Biosciences) by the enhanced chemiluminescent method (ECL Western blotting detection reagents and analysis system; Amersham Biosciences). The blots produced with lysates from tissues and cells were routinely stripped and reprobed for actin, using the mouse MAb JLA20 (VWR Deutschland, Darmstadt, Germany) to check for equal loading and transfer of proteins.
Processing of PRL by Purified Cath D, CL Tissue Extracts, and Culture Supernatant Concentrates In Vitro
PRL cleavage by purified Cath D. Human recombinant 23K PRL was produced in bacteria (pT7L-PRL vector) and purified as previously reported (61). Purified rat and bovine PRL were purchased from NHPP (Dr. A. F. Parlow, Harbor-UCLA Medical Center). Cleavage of PRL (150 ng) was performed with Cath D from bovine spleen (Sigma-Aldrich) in 100 µl of 20 mM citrate-phosphate buffer at various enzyme-to-substrate ratios. The pH was adjusted as needed by mixing the two buffers (0.2 M NaHPO4 and 0.1 M citric acid) in various amounts. BSA (100 µg) and/or the inhibitor pepstatin A (1.5 µM) were added when specified. Cleavage was allowed to proceed at 37°C for 3 h. Cleavage products were separated by SDS-PAGE and analyzed with anti-PRL antibodies by Western blotting.
PRL cleavage by tissue extracts. Shock-frozen CL pieces were pulverized with mortar and pestle, and cultured cells were scraped from the bottom of the flask. The samples were immediately transferred into 7.5 vol (wt/vol) ice-cold 0.25 M sucrose-0.1 M Tris·HCl (pH 7.4) buffer and homogenized with an Ultra Turrax T8 homogenizer (IKA Werke, Staufen, Germany). Subsequent successive 10-min centrifugations at 600 g, 3,300 g, and 25,000 g resulted in a high-speed pellet, which was washed twice. The pellet was weighed with an analytic balance, resuspended in reaction buffer (50 mM citrate phosphate-75 mM NaCl), and stored at –80°C. PRL (2.5 µg) was incubated with a 50-µg pellet in a final volume of 25 µl of reaction buffer at various pH values. The inhibitor pepstatin A (1 µl of a 1.5 µM solution) was added to some of the incubation tubes. Proteolysis was allowed to proceed at 37°C for 3 h. PRL fragments were electrophoresed, transferred to a nitrocellulose membrane, and analyzed by Western blotting.
PRL cleavage by culture supernatant concentrates. Culture supernatant concentrates were prepared as described above. Supernatant concentrate (2.5 µl) was mixed with 2.5 µl of PRL (1 µg/µl) in 20 µl of 50 mM citrate phosphate-75 mM NaCl buffer at various pH values. Pepstatin A (1 µl of a 1.5 µM solution) was selectively added to the reaction mix. After 3-h incubation at 37°C, the reaction mix was analyzed for the presence of PRL fragments by Western blotting.
Preparation of Lysosomal Fractions
CL tissue was pulverized in liquid nitrogen, transferred to 10 mM Tris·HCl, 0.3 M sucrose, and 1 mM EDTA (pH 7.5), and homogenized on ice with the Ultra Turrax homogenizer. This was followed by three successive centrifugations at 900 g (5 min), 3,000 g (10 min), and 10,000 g (25 min), respectively. The procedure resulted in pure lysosomal pellets, confirmed by the presence of electron-dense lysosomes observed by transmission electron microscopy in routinely fixed and embedded samples (not shown). The lysosomal pellet was considered to contain membrane "bound" lysosomal enzymes, whereas the resulting postlysosomal supernatant was considered to contain "free" lysosomal enzymes due to lysosomal fragility (47, 83). For Western blot analysis, the lysosomal pellet was further processed in lysis buffer (PBS, 0.2% Triton X-100, 20 mM EDTA, protease inhibitors; pH 5.0).
Detection of Enzymatic Activity
Total aspartic protease activity. Total aspartic protease activity was detected with the internally quenched fluorescent substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 [where MOCAc is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl; Calbiochem, Merck Biosciences, Darmstadt, Germany] (94). The supernatants from 12 wells of a 96-well plate were collected and pooled. The corresponding cell monolayers were treated with 200 µl of 0.9% Triton X-100 added to basal medium for 2 min at room temperature to disrupt the cell membranes. The cell lysates were collected and pooled, and cellular debris was removed by centrifuging at 1,200 g for 5 min. Supernatant and cell lysates were than concentrated with Centricon filter devices as described above. Subsequently, the 10-µl sample and substrate (20 µM) were incubated for 10 min at 40°C in 50 mM sodium acetate buffer, pH 4.0. The reaction was stopped by addition of 5% TCA, and the fluorescence of the intramolecularly cleaved fluorogenic substrate was measured in a monochromator (Molecular Devices, Ismaning/München, Germany). The excitation wavelength was set at 328 nm, and the emission was monitored at wavelength of 393 nm. Results expressed in relative fluorescence units (RFU) were determined by subtracting the RFU measured in the substrate mix alone from the RFU measured in the substrate mix plus supernatant. The experiment was repeated four times. The values obtained in the individual experiments were averaged and expressed as means with standard deviations.
Release of lactate dehydrogenase. The release of lactate dehydrogenase (LDH) was measured as an indicator of spontaneous cell lysis. LDH release was assayed with the CytoTox-ONE homogeneous membrane integrity kit (Promega). In this assay, 100 µl of culture supernatant concentrate or cell lysate were mixed with 100 µl of CytoTox-ONE reagent (substrate mix mixed 1:1 with assay buffer) and incubated at room temperature for 10 min. Stop solution was added. The excitation wavelength was set at 560 nm, and the emission was recorded at 590 nm to determine the generated fluorescent resorufin product.
Determination of the Influence of 16K PRL on Cell Number and Demonstration of Caspase-3 Cleavage
To determine whether 16K PRL alters proliferation of luteal EC, 10,000 cells were seeded per well of a 24-well culture plate. After 24 and 48 h, the cultures were exposed or not to bovine 23K PRL (500 ng/ml; NHPP) or to recombinant human 16K or 23K PRL (500 ng/ml). Generation of these recombinant molecules has been described in detail elsewhere (86). At 24-h intervals, the cells were trypsinized. Cells excluding Trypan blue were counted with a hemocytometer. The experiment was repeated three times. The values obtained in the individual experiments were averaged and expressed as means with standard deviation.
We assayed EC cultures for cleavage of caspase-3 into its active fragments. To this end, 375,000 EC were seeded per 75-cm2 flask; at confluency, the cultures were exposed or not for 48 h to bovine 23K PRL (500 ng/ml) or to recombinant human 16K or 23K PRL (500 ng/ml) or for 6 h to 200 nM staurosporine. At the end of the exposure, proteins were extracted from the cultures and analyzed by Western blotting with rabbit anti-cleaved caspase-3 MAb 5A1 (New England Biolabs, Frankfurt am Main, Germany), which detects endogenous levels of the large fragment (17K/19K) of activated caspase-3 resulting from cleavage adjacent to Asp175. Lysates of cytochrome c-treated or untreated Jurkat cells (New England Biolabs) were used, respectively, as positive and negative controls.
Reliability and Statistical Methods
Experiments were performed at least in triplicate. Only representative results are reported.
Statistically evaluated data are depicted as mean values ± SE. Statistical analyses were done by using Kruskal-Wallis one-way ANOVA on ranks followed by Tukey's test for pairwise comparisons. Statistical computations were performed with Sigma Stat (Systat Software, Erkrath, Germany). Differences are reported as statistically significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CL of bovine ovaries could be assigned to early (days 1–4 after ovulation), mid (days 5–17), and late (days 18–21) luteal stage by their macroscopic and histological appearance. A positive FVIII-related antigen labeling confirmed the cells with a cobble-stone morphology to be ECs. Abundant Nil red-positive droplets and high 3-hydroxysteroid dehydrogenase activity characterized flat GLC cultures (Fig. 1).
PRL is Synthesized in the Bovine CL
Although investigators have reported the presence of PRL mRNA in the bovine CL and in CL-derived cells (25, 74), it was necessary to check for the presence of PRL protein itself.
We first confirmed the presence of PRL mRNA in CL tissue (Fig. 2) and cells (EC and GLC, not shown). Amplification of the corresponding cDNA with PRL-specific primers yielded a single band corresponding to a PCR product of the expected length (525 bp), similar to the control RT-PCR bands obtained from bovine pituitary (Fig. 2) and GH4-C1 cells (not shown), both known as sources of PRL (22). Restriction with NcoI and RcaI yielded the expected digestion products, and sequencing of the PCR product cloned into pGEM-T revealed an exact match with the submitted nucleotide sequence of bos taurus PRL at GenBank (accession no. NM_173953, not shown). Although the RT-PCR analysis was not quantitative, the amplified PRL and internal control fragments appeared similar when the results of different CL estrous-cycle stages were compared.
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We next examined the variation of CL PRL and Cath D levels in the course of the bovine estrous cycle.
PRL was detected in the CL at all estrous cycle stages. The intensity of the 23K PRL band declined with respect to the actin band from early to late luteal stage (Fig. 3). Bands beneath the 23K band, likely to correspond to PRL fragments, showed only weak staining but became clearly visible when 100 µg of protein extract were loaded in the lanes. Blots probed for Cath D showed two intense bands, one at 44K and one at 30,000 (30K), corresponding, respectively, to the enzymatically active single-chain form and to the heavy chain of the enzymatically active two-chain form (60). In addition to these bands, we occasionally distinguished a very faint band above the 44K band, which may correspond to the inactive Cath D precursor procathepsin D (e.g., see Figs. 3 and 
5).
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Together, these Western blot data indicate that the Cath D protein content increases in the CL as the latter progresses toward the middle and end of its lifespan. Over the same period, the level of 23K PRL protein decreases. This could mean that 23K PRL becomes increasingly cleaved toward the end of the CL lifespan. We thus addressed the following questions: can Cath D cleave bovine PRL and can bovine CL tissue cleave it in a similar manner?
Cath D Processes Bovine PRL Into Multiple Fragments
Initially, NH2-terminal 14K and 16K PRL fragments were reported to result from Cath D cleavage of rat PRL (4), but, subsequently, human PRL was reported to be resistant to Cath D cleavage (46). Although this was disproved by a later study (66), we found it necessary to test whether bovine PRL is cleaved by Cath D.
When bovine PRL was incubated with bovine Cath D at a 1:100 enzyme-to-substrate ratio for 3 h, the 23K hormone was cleaved into 14K and 16K fragments under acidic conditions (Fig. 4, bottom left). Incubation of rat or human 23K PRL with bovine Cath D at the same enzyme-to-substrate ratio yielded only a 16K fragment (Fig. 4, top and middle left). Cleavage was blocked when the pH was raised to 7 and when the specific Cath D inhibitor pepstatin A was added to the reaction mixture. The optimal pH for cleavage was pH 3, and the presence of BSA in the digestion mixture facilitated cleavage at a higher pH, up to pH 5.5 (not shown). In similar experiments (Fig. 4, right), the enzyme-to-substrate ratio was varied. At the highest ratio tested, the major band observed corresponds to an apparent molecular weight of 14K. Between the ratios of 1:20 and 1:100, both 16K and 14K fragments were detected. Additional fragments of higher relative molecular weight, such as 21,000, were noticed in acidic preparations but were considered nonspecific because they occurred independently of the presence or absence of pepstatin A and of any source of Cath D (Fig. 4 and also Figs. 5 and 7). The 16K and 14K fragments appeared to be NH2-terminal fragments because they produced a signal when probed with anti-PRL MAbs (MAb 5G2 and MAb 4C10) recognizing an epitope near the NH2 terminus (not shown). It emerges from these results that bovine Cath D cleaves bovine PRL even more efficiently than it cleaves rat and human PRL.
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Tissue homogenates from mammary gland (14), liver (14), and prostate (16) have been shown to process PRL into 16K fragments in vitro. Because full-length PRL declines in the CL between the early and late luteal stages, we wondered whether this decline reflected the processing of PRL in the CL. We therefore tested whether bovine CL tissue extracts were able to cleave PRL in vitro.
Bovine 23K PRL was incubated at acidic pH with CL tissue extracts corresponding to different estrous cycle stages. Western blot analysis of the reaction mixture revealed that the extracts could generate cleaved PRL forms (Fig. 5). The two major bands beneath that corresponding to 23K PRL, and showing estimated molecular weights of 16K and 14K, were comparable to those produced by Cath D-mediated proteolysis in vitro. Proteolysis was maximal under acidic conditions and was blocked by the Cath D inhibitor pepstatin A. No differences in cleavage activity were noted between CL extracts corresponding to different estrous cycle stages (not shown). These results strongly suggest that PRL is cleaved in bovine CL tissue and that Cath D may affect this cleavage.
Cultured Bovine CL-Derived Cells Synthesize and Release Active Forms of Cath D
We next focused on cultured luteal EC and GLC to see whether these cells can produce and secrete Cath D.
Cell lysates and concentrated conditioned media (Fig. 6) revealed the presence of two major bands, of 44K and 30K, when probed with anti-Cath D. These molecular weights match those of the active Cath D single-chain form and the heavy chain of the active Cath D two-chain form, respectively (60). The presence of this aspartic protease in the cell supernatant was further confirmed by testing the ability of culture supernatants to quench a fluorescent Cath D substrate. Both media from EC and GLC cultures displayed Cath D activity clearly above the basal level. They also displayed LDH activity above the basal level, but this activity was comparatively much lower than if staurosporine-induced enzyme release had occurred, due to membrane leakage in the late phase of staurosporine-induced apoptosis (1, 45).
16K PRL is Generated by Culture Medium Conditioned by CL Cells
We next tested whether the media of luteal EC and GLC cultures have the ability to generate PRL fragments.
Exogenous bovine PRL added to acidified nonconditioned medium incubated at 37°C for 3 h was detectable as a major 23K and a minor 21K band. In contrast, exogenous bovine PRL incubated similarly in media conditioned by CL-derived cells gave rise to 16K and 14K PRL fragments (Fig. 7). The fragments migrated like the fragments generated in vitro by Cath D-mediated proteolysis. The fragments were not detected when pepstatin A was added to the conditioned medium or when the incubation was performed at pH 7.
Recombinant 16K PRL Inhibits Proliferation of CL-Derived EC
To determine whether PRL cleavage has an effect on luteal EC, we exposed cultures of these cells to recombinant human 16K PRL. Cultures exposed to 16K PRL showed a significantly reduced growth rate compared with unexposed cultures, as early as 8 h after the start of treatment (Fig. 8, top).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Data on PRL-receptor knockout mice (37) and from experiments with the antiangiogenic 16K PRL fragment point to a new role for PRL signaling in the CL: 23K PRL signaling may be required to promote CL vascularization, whereas its bioactive fragments are likely to favor CL regression. Thus the balance between 23K and 16K PRL might determine whether the CL persists and develops further to maintain pregnancy or regresses and allows the development of a new ovulatory follicle.
PRL Synthesis in the CL
In this study, we provide evidence that luteal EC and GLC, two major cell types of the bovine CL, synthesize and secrete PRL. This confirms and extends results of very recent studies conducted by our group (70) and by Shibaya et al. (74), revealing the presence of PRL and PRL-receptor mRNA in the bovine CL. Local PRL synthesis and secretion raise the question of the role that the hormone may play in the bovine CL. Because the bovine cell types studied here express both the long and short forms of the PRL receptor (70, 74) and because treatment with receptor-neutralizing antibodies inhibits the growth of these cells in culture (70, 74), autocrine or paracrine effects of PRL are hypothesized.
PRL and PRL Fragments in the CL
We also show that full-length 23K PRL drops in the bovine CL to almost undetectable levels toward the end of the CL lifespan. This might be caused by decreased local PRL supply, decreased local synthesis, and/or increased generation of PRL fragments. Reduced synthesis is in line with the reduced PRL mRNA level observed at the end of the CL lifespan (74); enhanced proteolysis is suggested by two findings presented here: the fact that the Cath D rises as the 23K PRL level falls and the ability of Cath D to cleave 23K PRL in a manner similar to that reported for rat and human PRL (4, 66). We further show that CL tissue extracts can cleave PRL under acidic conditions. The CL is thus equipped for partial PRL proteolysis.
Increased synthesis of hydrolytic enzymes like Cath D and their release from lysosomes are known to occur in many instances of cell destruction (20). They also herald CL regression. At the onset of luteal regression, autophagic vacuoles and lysosomes increase in number and size (23), and we and others observe an increase in lysosomal-bound and free Cath D. PGF2 is proposed to affect the lysosomal membrane, causing the release of bound Cath D into the cytosol (47, 67). Both autophagosomes-autophagolysosomes and the Cath D free in the cytosol may initiate cell death without (88) and with caspase involvement (43), respectively.
The ideal way to demonstrate PRL cleavage at the end of the CL lifespan would be to detect specific cleavage products in the regressing CL. These products appear to be short lived and transiently appear in earlier stages. However, as suggested by the progressive degradation of bovine PRL at high enzyme-to-substrate ratios, it is also imaginable that Cath D might act in concert with other proteases (52) or activate other proteases present in the CL (58), either directly or indirectly (76, 89), causing the fragments to be further processed.
Lysosomes and Cath D Exocytosis in the CL
In addition to cell-associated Cath D, secreted or released Cath D might play a role in the CL. We detected the presence of 44K and 30K molecular weight Cath D forms from either endosomes or lysosomes in supernatants of cultured CL-derived cells. This contrasts with most of the established literature, which have suggested that, in mammalian cell cultures, only the inactive precursor procathepsin D is secreted (90). However, the secretion of mature Cath D forms active on native PRL has been shown to occur in cultured rat mammary epithelial cells (15). Nonetheless, we cannot exclude that, in our cultures, small amounts of Cath D could arise from spontaneous cytolysis after normal cell shedding, since LDH activity also slightly increased over time in the supernatants.
Procathepsin D is reported to require an acidic pH to be proteolytically processed to the active intermediate 44K single-chain and to the more stable 30K and 14K two-chain form (39). In this study, we demonstrate the presence of the 44K and 30K Cath D forms in culture supernatants, in keeping with the finding that Cath D can cleave PRL to 16K PRL outside the cell compartment (15, 16). Yet only at acidic pH do our supernatants display Cath D-like enzymatic activity that can cleave native 23K PRL to 16K PRL. PRL cleavage activity is highest at pH 3 and disappears at pH 5.5. This is in agreement with the observation that an acidic pH is required for Cath D activity in vitro (12, 55, 91). However, assays in vitro have shown that Cath D is stable between pH 1 and 9 (44) and that it can cleave some substrates at pH 5.5 (44). Furthermore, aggregate or spheroid cultures, instead of a two-dimensional culture setup, may allow a more physiological pH for PRL cleavage (5). We have noted PRL cleavage up to pH 5.5 after addition of BSA, in keeping with the finding that physiological activators may facilitate Cath D cleavage at a more basic pH (50).
Hypoxia, Glycolysis, and Acidity in the CL
In solid tumors, the extracellular environment where Cath D is suggested to occur (50, 76, 91) is known to be continuously acidic (36) because of hypoxia (40), changes in energy metabolism (30), and a slow rate of exchange between tissue and body fluids. Hypovascularity and anaerobic glycolysis are observed in the ovary as well, during late folliculogenesis and early CL development (11, 56). At these times, the extracellular microenvironment resembles that of solid tumors (9, 56). Mathematical models (35) and intrafollicular oxygen measurements suggest that mature follicles are heavily underoxygenated. One study showed that the PO2 level in follicular fluid falls from 90 to 50–60 Torr (26) at this time, when glucose is preferentially converted to lactic acid.
Hypoxia and its metabolic consequences again prevail when the CL is regressing. Vasoconstriction, detachment of EC, and occlusion of blood vessels are early events of CL regression (54, 57, 59). The breakdown of blood vessels results in a diminished oxygen supply to the luteal parenchyma. When oxygen tension is experimentally reduced in the media of CL from pseudopregnant rats, lactic acid accumulates (29). Acidification of luteal cells leads to hydrolase release and cell death due to activation of p53- and caspase-3-dependent apoptosis pathways, autophagy, and loss of function of critical pH-sensitive genes (77). Protein H+-pumps, which actively extrude H+ in exchange for extracellular cations, are present and active in EC (2, 71) and macrophages (84) populating the CL. Their role in pH homeostasis in the CL requires further investigation. In addition, it is interesting to note that short periods of hypoxia may stimulate the exocytosis of mature lysosomal enzymes, among which is Cath D, as it has been shown to occur in hepatocytes (13).
On the basis of the above findings, an acidic extracellular environment allowing PRL cleavage by Cath D appears likely in the regressing CL. Cath D might also perform functions independent of this protease activity. For instance, it might recruit other proteases, such as cathepsin L in the CL (58), by binding to their natural inhibitors (48) or it might act via a putative activation peptide receptor (90) to stimulate the proliferation, motility, and survival of certain CL cells, such as fibroblasts (48) and EC (7). At present, we cannot exclude that part of the aspartic proteolytic capacity observed in the ovary is due to cathepsin E (Cath E) and not to Cath D. Cath E has enzymatic properties similar to Cath D [e.g., susceptibility to pepstatin A, acidic pH optimum (92)], and MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 is a substrate for both proteases (95). Cath E is of nonlysosomal origin and not secretory (87). Cath E is retained within vesicular structures mainly associated with the endoplasmatic-endosome compartment. Its tissue and cellular distribution is more restricted than the one of Cath D (72). It is of note that particular cells of the immune system such as macrophages and lymphocytes contain Cath E. Whether PRL is a Cath E target and whether Cath E is present in the CL is presently unknown.
16K PRL Affects CL-Derived EC
We finally show that 16K PRL, the product of PRL cleavage by Cath D, inhibits the growth of CL-derived EC and is thus likely to affect CL vascularization in vivo. This growth inhibition correlates with caspase-3 activation. Such a correlation has been described in the literature (86) and is in line with the observation that 16K PRL promotes apoptosis-induced vascular regression (24). It may well be that 16K PRL exerts further effects on positive and negative regulators of cell cycle progression in the CL (85), but this is beyond the scope of the present paper.
Conclusions
In conclusion, our study demonstrates that the bovine CL is not only a PRL target organ but also a site of PRL synthesis. We further show that CL-derived cells deliver Cath D to the extracellular space and that this protease can cleave PRL into bioactive fragments affecting CL-derived EC. Therefore, given the importance of vascular changes in the course of the estrous cycle, we propose that the balance between 23K and 16K PRL in the CL might influence whether it persists and develops for the maintenance of pregnancy or regresses and allows the development of a new preovulatory follicle. We are only beginning to appreciate the influence of the microenvironment on CL biology. An understanding of how the microenvironment determines the generation of bioactive fragments should prove useful not only in reproductive biology but also in other fields, such as regenerative medicine, therapeutic angiogenesis, and tissue remodeling.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* S. Erdmann and A. Ricken contributed equally to this work. 
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