Obesity-associated dysregulation of calpastatin and MMP-15 in adipose-derived stromal cells results in their enhanced invasion R1

Materials

Anti-CD45-PeCy7, anti-CD11b-PeCy5, anti-CD166-PE, anti-CD105-PE, anti-CD90-PeCy5, anti-CD34- PE, isotype-control FITC human IgG1 and isotype-control PE human IgG2a were purchased from Beckman Coulter (Indianapolis, IN). Anti-CD44-APC was purchased from BD Biosciences (San Jose, CA). Anti-MMP-12, anti-MMP-15, anti-MMP-21, anti-MMP-23, anti-MMP-28, anti-TIMP-4, anti-calpain-1, anti-calpain-2, anti-calpain-3, anti-calpain-4, anti-calpain-5, anti-calpain-6, anti-calpain-7, and anti-calpain-10 were purchased from Abcam (Cambridge, MA). Anti-calpastatin and anti-actin were purchased from Sigma (St. Louis, MO). Rabbit anti-mouse and goat anti-rabbit HRP-conjugated secondary antibodies were purchased from Abcam. Aspartate proteinase inhibitor, pepstatin A (100 mM), serine proteinase inhibitor, soybean trypsin inhibitor, (SBTI; 100 μg/mL), and cysteine proteinase inhibitor, E64d (1 μM), were purchased from Sigma. Synthetic matrix metalloproteinase (MMP) inhibitor, GM6001 (25 μM), was purchased from Millipore (Bedford, MA), and cathepsin inhibitor, 1,3-Bis(CBZ-Leu-NH)-2-propanone (10 μM), was purchased from EMD Chemicals (Gibbstown, NJ). The calpain inhibitor, acetyl-calpastatin (0.2 μM), was purchased from AnaSpec (Fremont, CA), and the caspase inhibitor, Z-VAD-FMK (20 μM), was purchased from Promega (Madison, WI).

Human Subjects

Primary human adipose stem cells (ASCs) were obtained from 24 Caucasian females undergoing elective liposuction procedures, as previously described.¹² ASCs were isolated from processed lipoaspirates from the subcutaneous abdominal adipose tissue of obese (Ob⁺Ab⁺) or non-obese (Ob⁻Ab⁺) subjects and from non-abdominal subcutaneous adipose depots of obese (Ob⁺Ab⁻) and non-obese (Ob⁻Ab⁻) subjects. Additional demographic information can be found in Supplemental Material 1. No statistical significance in age was observed between the groups. Six donors per group were individually characterized and expanded. All protocols were reviewed and approved by the Pennington Biomedical Research Center Institutional Review Board and all human participants provided written informed consent.

Cell Culture

Conditioned Media

MDA-MB-231 cells were cultured in DMEM, supplemented with 10% FBS, 100 units per mL penicillin, and 100 μg/mL streptomycin for 24 hours. After 24 hours, the cells were washed twice with serum free DMEM and further cultured for 48 hours in serum free DMEM. The conditioned media was filtered (0.22 μM) to remove any cellular debris.

Differentiation Protocols

Colony Forming Unit Assay

ASCs were plated at a density of 100 cells on a 10 cm² plate in CCM and incubated for 14 days. Plates were rinsed three times with PBS, and 10 mL of 3% crystal violet (Sigma) was added for 30 minutes at room temperature. Plates were washed three times with PBS and once with tap water. Colonies that were larger than 2 mm in diameter were counted. Each experiment was performed in triplicate.

Flow Cytometry

ASCs were harvested with 0.25% trypsin/1mM EDTA for 3-4 minutes at 37°C. A total of 3 × 10⁵ cells were concentrated by centrifugation at 500 × g for 5 min, suspended in 50 μl PBS and labeled with the primary antibodies. The samples were incubated for 30 minutes at room temperature and washed three times with PBS. The samples were then analyzed by FACScan (FACScalibur; BD Biosciences) with CellQuest software (BD Biosciences). To assay cells by forward and side scatter of light, FACScan was standardized with microbeads (Dynosphere uniform microspheres; Bangs Laboratories Inc.; Thermo Scientific; Waltham, MA). At least 10,000 events were analyzed and compared with isotype controls.

Transwell Invasion Assay

Invasion assays were performed in transwell inserts with 8-μm pore membrane filters pre-coated with a growth factor-reduced Matrigel layer to mimic basement membranes (BD Biosciences). ASCs were grown to 70% confluent prior to harvesting by trypsinization and pooled based on obesity status and depot site. ASCs were loaded (5 × 10⁴ cells/well in 500 μL) onto the upper chamber, while 750 μL MDA-MB-231 conditioned media was loaded onto the bottom chamber. Where indicated, ASCs were pretreated with protease inhibitors for 4 hours prior to loading onto the upper chamber, and fresh protease inhibitors were added to the upper chamber for the duration of the invasion assay. After overnight incubation, the lower side of the transwell insert was carefully washed with cold PBS and non-invading cells remaining on the top chamber were removed with a cotton tip applicator. Invading cells were stained with Calcein-AM (2 μg/ml; Invitrogen; Grand Island, NY) and measured on a fluorescent plate reader (FLUOstar optima; BMG Labtech Inc.; Durham, NC). Data was normalized by dividing the values obtained with or without MDA-MB-231 conditioned media as a chemoattractant by the values obtained without conditioned media. Where ASCs were treated with protease inhibitors, the data was normalized by dividing the values obtained with each treatment group by the values obtained without protease inhibitor treatment to determine the percentage of inhibition.

RNA Isolation and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Subconfluent cultures of ASCs were grown in conditioned media for 24 hours. The three donors in each category were pooled based on obesity status and depot site prior to total RNA extraction from ASCs using TRIzol reagent (Invitrogen) and purification with DNase I digestion (Invitrogen). A total of 200 ng of cellular RNA was used for cDNA synthesis and PCR with the SuperScript III One-Step RT-PCR System with Platinum Taq (Invitrogen). No-template controls (negative control, neg ctrl) were run to rule out cross contamination of reagents and surfaces. All PCR products were analyzed by gel electrophoresis on 2.0% agarose gels with ethidium bromide stain. Gels were imaged with ImageQuant LAS 4000 (GE Healthcare Life Science; Piscataway, NJ) and representative image of one of three independent experiments are shown. Quantitative analysis of RT-PCR was performed by densitometry. MMP primer sets were designed by Kohrmann et al.¹⁴ The primer sets used in the RT-PCR are listed in Supplemental Material 2.

Protein Isolation and Western Blot

Subconfluent cultures of ASCs were grown in conditioned media for 24 hours. The three donors in each category were pooled based on obesity status and depot site prior to cell lysis with RIPA buffer (Pierce; Thermo Scientific). The cell lysate was clarified by centrifugation at 15,000 × g for 15 min. Protein concentration was determined by the BCA Protein Assay (Pierce). Lysate (20 μg) was resolved on 4-12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Invitrogen). Blots were blocked with blØk Noise Canceling Reagents (Millipore). Blots were then incubated with the primary antibodies overnight at 4°C and washed with PBS with Tween 20 three times before incubated with species-specific IgG conjugated to horseradish peroxidase for 1 hour at room temperature. Antigen-antibody complexes were visualized after incubation in chemiluminescence reagent (Invitrogen). Blots were imaged on an ImageQuant LAS 4000 and representative blots of one of three independent experiments are shown. Quantitative analysis of Western blots was performed by densitometry.

Zymography

Subconfluent cultures of ASCs were grown in conditioned media for 24 hours. The three donors in each category were pooled based on obesity status and depot site prior to suspension media collection and concentrated with protein concentrators (Pierce). A total of 20 μg of protein was loaded under non-denaturing conditions into Novex Precast Polyacrylamide Zymography Gels supplemented with 0.1% gelatin (Invitrogen). Electrophoresis was performed at a constant voltage of 120 V for 90 min. Gels were washed with Novex Zymogram Renaturing Buffer (Invitrogen) and incubated overnight in Novex Zymogram Developing Buffer (Invitrogen) according to Manufacturer’s instructions. Gels were stained with Simply Blue SafeStain (Invitrogen) for 1 hour and then washed with water for 1 hour. Gels were incubated with Gel-Dry Drying Solution (Invitrogen) for 5 minutes before being placed between cellophane for drying overnight. After gels dried overnight, zymograms were recorded with ImageQuant LAS 4000 and representative zymographs of one of three independent experiments are shown. Quantitative analyses of zymographs were analyzed by densitometry.

Stable Transfection of shRNA

Short hairpin RNA constructs targeting MMP-14, MMP-15, calpain-4, and calpastatin and an shRNA construct targeting a non-human gene serving as a control were purchased from SABiosciences (Frederick, MD). The following shRNA constructs were used: MMP-14 (5′-gcccaatggaaagacctactt-3′), MMP-15 (5′-aaccgcgtcctggacaactat-3′), calpain-4 (5′-cgacgctactcagatgaaagt-3′), calpastatin (5′-aaaccacaagacatgatttct-3′), and non-coding gene (5′-ggaatctcattcgatgcatac-3′). ASCs were transfected with shRNA using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and 3 pulses. Cells were allowed to recover for 48 hours before selection with Geneticin (Invitrogen). For selection, 600 μg/mL of Geneticin diluted in fresh CCM was replaced every 2 days, and the selection was performed for 14 days.

Chick Chorioallantoic Membrane (CAM) Assays

Fertilized white leghorn chick embryos were obtained from Charles River Laboratories (Wilmington, MA). Following 2 days of incubation in a 37°C humidified incubator, eggs were swabbed with 70% ethanol, and embryos were carefully removed from the shell using a Dremel tool and placed in a sterile, covered weigh boat and returned to the incubator. On day 10 of incubation, 10⁶ ASCs were labeled with 10 μM CellTracker Green CMFDA (Invitrogen), resuspended in 100 μl PBS, and added onto the CAM on a nylon mesh for localization of the cells as previously described.^15-18 After 3 days of incubation, the areas of interest were dissected out, fixed in 4% paraformaldehyde, frozen in OCT, and cut into 8 μm sections. The percentage of invading cells was quantified in three or more randomly selected fields. Depth of invasion from the CAM surface was defined as the leading front of three or more invading cells in randomly selected fields. Images were acquired on Olympus BXS1W1 spinning disk confocal microscope (Center Valley, PA) with Hamamatsu EM-CCD C9100 camera (Hamamatsu City, Japan) using Slidebook version 5.0 software (Olympus).

Statistical Analysis

All values are presented as means ± SD. The statistical differences among two or more groups were determined by ANOVA, followed by post-hoc Dunnet multiple comparison tests versus the respective control group. The statistical differences between two groups were performed by Student’s t-test. Statistical significant was set at P < .05. Analysis was performed using Prism (Graphpad Software, San Diego, CA).

Characterization of ASCs isolated from donors based on obesity status and depot site of origin

ASCs were isolated from processed lipoaspirates of obese (Ob⁺) and non-obese (Ob⁻) subjects undergoing elective plastic surgery and obtained from the subcutaneous abdominal adipose tissue (Ab⁺) or non-abdominal subcutaneous depots (Ab⁻). The ASC from all groups (Ob⁺Ab⁺, Ob⁺Ab⁻, Ob⁻Ab⁺, Ob⁻Ab⁻) were found to be positive for CD44, CD90, CD105, and CD166 and negative for CD34, CD45, and CD11b via flow cytometry (Figure 1A). Each group of ASCs was able to differentiate into adipocytes and osteoblasts (Figure 1B) as well as generate colony-forming units (Figure 1C). No differences were observed among the four groups for ASC differentiation or self-renewal capacity as defined by colony forming units (Figure 1C).

Ob⁺Ab⁺ ASCs demonstrate increased invasion towards breast cancer cell conditioned media

To investigate the invasive potential of ASCs in response to breast cancer cells, conditioned media from MDA-MB-231 cells was placed in the lower chamber of a growth factor reduced Matrigel coated transwell insert, while ASCs were plated onto the top chamber. All four ASC groups invaded towards conditioned media with statistically significance increases compared to control after 24 hour (P<0.05). Invasion of Ob⁺Ab⁺ ASCs was increased by 7.5 fold relative to control (unconditioned media), whereas the invasion of Ob⁺Ab⁻, Ob⁻Ab⁺, and Ob⁻Ab⁻ ASCs was increased by 2.8-, 3.6-, and 3.8-fold, respectively. Invasion of Ob⁺Ab⁺ ASCs invaded was significantly more compared to Ob⁺Ab⁻, Ob⁻Ab⁺, and Ob⁻Ab⁻ ASCs (P<.001)

GM6001 and acetyl-calpastatin inhibit Ob⁺Ab⁺ ASC invasion

To determine the protease responsible for the robust invasion of Ob⁺Ab⁺ ASCs towards breast cancer conditioned media, all four groups of ASCs were pretreated with blanket inhibitors for 4 hours to the four protease classes found in humans: cysteine, serine, aspartate, and matrix metalloproteinases (MMP). Inhibition of Ob⁺Ab⁺ ASCs with E64d and GM6001 significantly impaired the invasion of these ASCs by 34% and 62%, respectively, when compared with untreated control cells (Figure 3A). To further reveal the protease(s) involved, Ob⁺Ab⁺ ASCs were pretreated with blanket inhibitors to the three classes of cysteine proteases: cathespins, calpain, and caspases. Pretreatment of the ASCs with calpain inhibitor acetyl-calpastatin reduced the invasion of Ob⁺Ab⁺ ASC by 40% relative to cells that were not treated (Figure 3B). ASCs pretreated with cathespin and caspase inhibitors had no effect on invasion. No changes in cell viability or proliferation were observed after treatment with protease inhibitors (data not shown). These experiments indicate that both MMPs and calpains are involved in the invasion of Ob⁺Ab⁺ ASCs.

mRNA expression of MMPs and calpains vary among the four groups

To further identify the protease(s) involved in the invasion, ASCs were grown in the absence and presence of MDA-MB-231 conditioned media prior to the isolation of RNA and detection of mRNA expression by RT-PCR. Primer sets were constructed for the detection of specific MMPs and calpains along with the endogenous inhibitors of these proteases, which included the TIMPs and calpastatin, respectively (Supplemental Material 2).

Of the MMPs and TIMPs, the mRNAs for 18 MMPs and 4 TIMPs were detected in all groups of ASCs (Supplemental Material 3). However, the levels of mRNA expression of MMP-2, -9, -12, -15, -21, -23, and -28 as well as TIMP-4 varied among the four groups (Figure 4). More specifically, levels of MMP-2, MMP-9, and MMP-28 were increased by 1.3-fold, 1.8-fold, and 1.8-fold, respectively, in Ob⁺ ASCs. In contrast, the mRNA level of MMP-12, MMP-21, MMP-23 and TIMP-4 were increased by 1.3-fold, 2.2-fold, 2.4-fold, and 1.4-fold, respectively, in Ob⁻ ASCs. Of particular interest was the increase in MMP-15 expression in the Ob⁺Ab⁺ ASCs compared to the other 3 groups of ASCs, indicating the potential involvement of MMP-15 in the enhanced invasion of Ob⁺Ab⁺ ASC.

Of the 15 calpains and calpastatin, the mRNAs for 9 calpains and calpastatin were present at detectable levels in all groups of ASCs (Supplemental Material 3; Supplemental Material 4); however, levels of calpain-4, -6, -7, and -10 varied among the four groups (Figure 4; Supplemental Material 4). mRNA expression of calpain-4 was increased 2.5-fold in the Ob⁺Ab⁺ ASCs compared to the other three groups. RNA expression of calpain-6 and calpain-7 was decreased in the Ob⁺ groups by 2.3-fold and 2.5-fold, respectively. In addition, Ob⁺Ab⁺ ASCs showed significant reduction by 3.5-fold in calpastatin mRNA expression (Figure 4; Supplemental Material 4).

Expression and activity of MMPs and calpains vary among the four groups

Western blots or zymograms were performed on the protease or protease inhibitors that displayed varying RNA expression among the four groups. To confirm the protein expression of MMP-2 and MMP-9, zymography was performed with conditioned media serving as a baseline control. MMP-2 and MMP-9 activity levels were increased in Ob⁺ groups as compared to Ob⁻ groups (Figure 5A).

Western Blotting was performed for MMP-12, MMP-15, MMP-21, MMP-23, MMP-28, TIMP-4, calpain-4, calpain-6, calpain-7, calpain-10, calpastatin, with beta-actin serving as a control. The expression of MMP-14 was assessed as well because previous studies have suggested MMP-14 as the sole molecule responsible for the invasion of bone marrow derived MSC (BMSCs).^{17, 19} Western blot analysis showed no differences in protein expression of MMP-12, MMP-14, MMP-21, MMP-23, MMP-28, TIMP-4, calpain-6, calpain-7, or calpain-10. Western Blot confirmed the decrease in calpastatin expression by 4.0-fold in Ob⁺Ab⁺ ASCs relative to other ASCs (Figure 5B). Increased expression in MMP-15 and calpain-4 by 2.2- and 2.0-fold, respectively, was observed in Ob⁺Ab⁺ ASCs when compared with the other ASC groups (Figure 5B).

Reduced calpastatin and enhanced calpain-4 expression increase invasion

To confirm the functional role of calpastatin and calpain-4 in invasion, ASCs were transfected with calpastatin or calpain-4 shRNA (Figure 6A). Knockdown efficiency was assessed with Western blot analysis after antibiotic selection was complete. Calpastatin knockdowns in Ob⁻Ab⁻, Ob⁻Ab⁺, and Ob⁺Ab⁻ ASCs showed a reduction in protein expression of calpastatin by 78%, 82%, and 80%, respectively. Calpastatin knockdown in Ob⁺Ab⁺ ASCs was comparable to nontransfected ASCs, suggesting knockdown efficiency of calpastatin in Ob⁺Ab⁺ was low, possibly due to low levels of endogenous calpastatin in these cells. Protein expression of calpain-4 was decreased in all four groups after shRNA transfection and antibiotic selection, with the greatest decrease of 87% observed in Ob⁺Ab⁺ ASCs.

Functionally, Ob⁻Ab⁻, Ob⁻Ab⁺, and Ob⁺Ab⁻ ASCs transfected with calpastatin shRNA demonstrated increased invasion towards conditioned media compared to shRNA negative control cells or naïve untransfected cells (Figure 6B). Invasion of Ob⁺Ab⁺ ASCs transfected with calpastatin shRNA showed a similar invasive phenotype as the naïve untransfected cells or shRNA negative control Ob⁺Ab⁺ ASCs (Figure 6B). No significant difference in invasion was observed between the four groups of ASCs after calpastatin shRNA transfection. These experiments confirm that low calpastatin expression correlates with enhanced invasion. Calpain-4 knockdowns in Ob⁺Ab⁺ ASCs diminished the invasion by 5-fold and negated the enhanced invasion observed in Ob⁺Ab⁺ ASCs exposed to breast cancer cell conditioned media. These observations indicate that the simultaneous reduction in calpastatin and increase in calpain-4 in Ob⁺Ab⁺ ASCs contribute to the cellular invasion.

Assessing variability of MMP-15, calpain-4, and calpastatin expression

In order to assess the variability between donor samples within groups and between groups, an additional 3 donors were obtained for each ASC group, resulting in a total of 24 donor samples. ASCs were primed in MDA-MB-231 conditioned media prior to the isolation of protein and, where indicated, were pooled according to donor’s obesity status and depot site, prior to blotting with MMP-15, calpain-4, or calpastatin antibody. Quantification of Western blots with densitometry demonstrated no statistically significant variability within the groups, while statistically significant differences were observed between the groups as demonstrated with the analysis of the pooled samples (Supplemental Material 5).

Inhibition of MMP-15 shRNA reduced invasion of ASCs

To determine the role of MMP-15 on ASC invasion, ASCs were transfected with MMP-15 shRNA followed by antibiotic selection. MMP-14 shRNA was also assessed because previous studies have suggested that MMP-14 may be responsible for BMSC invasion.^10,11 Knockdown efficiency was assessed through Western blot analysis. Protein expression of MMP-14 and MMP-15 was decreased in all four groups after shRNA transfection and antibiotic selection (Figure 6A). Since high levels of endogenous MMP-15 were found in Ob⁺Ab⁺ ASCs, the greatest decrease in expression after shRNA knockdown was observed in Ob⁺Ab⁺ ASCs.

Functionally, inhibition of MMP-14 resulted in a 2-fold decrease in all four groups, indicating that all ASCs can utilize, but do not require MMP-14 to invade (Figure 6B). After MMP-14 shRNA knockdown, Ob⁺Ab⁺ ASCs retained their enhanced invasion, suggesting that MMP-14 is not responsible for the enhanced invasion of Ob⁺Ab⁺. In contrast, inhibition of MMP-15 demonstrated reduced invasion in Ob⁺Ab⁺ ASCs by 2-fold, although no significant changes were observed in the invasion of Ob⁺Ab⁻, Ob⁻Ab⁺, or Ob⁻Ab⁻ ASCs. ShRNA knockdown of MMP-15 abolished the enhanced invasion of Ob⁺Ab⁺ ASCs in response to breast cancer cell conditioned media, suggesting that these cells utilize an alternative pathway with MMP-15 as their central player.

Invasion of hASCs in vivo

To evaluate ASC invasion in a complex ECM model system that inherently contains various chemokines and chemoattractants, an in vivo chick CAM model was used. The CAM contains a type I collagen-rich ECM barrier commonly used to study invasive processes.^15,18 The shRNA negative control of Ob⁺Ab⁺ ASCs demonstrated enhanced invasion compared to control Ob⁺Ab⁻, Ob⁻Ab⁺, or Ob⁻Ab⁻ ASCs. While MMP-14 knockdown in ASCs significantly reduced the invasion of Ob⁺Ab⁻, Ob⁻Ab⁺, and Ob⁻Ab⁻ ASCs, Ob⁺Ab⁺ ASCs maintained their more invasive phenotype compared to the other groups. In contrast, knockdowns of MMP-15 and calpain-4 in Ob⁺Ab⁺ ASCs reduced the invasive phenotype of these cells to the same invasiveness as other ASCs, while knockdowns of MMP-15 and calpain-4 in Ob⁺Ab⁻, Ob⁻Ab⁺, and Ob⁻Ab⁻ ASCs had no effect on the invasion of these cells, suggesting the use of MMP-15 and calpain-4 by Ob⁺Ab⁺ ASCs but not by Ob⁺Ab⁻, Ob⁻Ab⁺, or Ob⁻Ab⁻ ASCs. In comparison, knockdowns of calpastatin in Ob⁺Ab⁺ ASCs demonstrated no effect on the invasion of these cells, while knockdowns of calpastatin in Ob⁺Ab⁻, Ob⁻Ab⁺, and Ob⁻Ab⁻ ASCs demonstrated enhanced invasion. Interestingly, although levels of invasion in Ob⁺Ab⁻, Ob⁻Ab⁺, and Ob⁻Ab⁻ ASCs were increased with calpastatin shRNA, naïve Ob⁺Ab⁺ ASCs remained more invasive, indicating that an additional molecule may be required for invasion.