Identification of regulatory Hck and PAI-2 proteins in the monocyte response to PEG-containing matrices

Abstract

Mass spectrometry is a powerful proteomic tool enabling researchers to survey the global proteome of a cell. This technique has only recently been employed to investigate cell-material interactions. We had previously identified material scarcity and limited adherent cells as challenges facing mass spectrometric analysis of cell-material interactions. U937 adherent to tissue culture poly(styrene) was used as a model system for identifying proteins expressed by adherent monocytes and analyzed by HPLC coupled offline to MALDI-ToF/ToF (LC-MALDI). We identified 645 proteins from two cation fractions of crude U937 monocyte cell lysate. Forty three proteins of interest from the 645 were chosen based on literature searches for relevance to monocyte-material inflammation and wound healing. Proteins such as 40S ribosomal protein S19 and tyrosyl tRNA synthetase highlight the ability of LC-MALDI to identify proteins relevant to monocyte-material interactions that are currently unexplored. We used PEG-based semi-interpenetrating polymer networks and PEG-only hydrogels to investigate surface dependent effects on the Src family kinase Hck and plasminogen activator inhibitor-2 (PAI-2) using the pyrazolo pyrimidine small molecule inhibitor PP2 and exogenous urokinase plasminogen activator addition, respectively. Hck is well researched in cell adhesion while PAI-2 is virtually unknown in cell-material interactions. U937 on TCPS and PEG-only hydrogels secreted similar levels of inflammatory cytokines and gelatinase MMP-9. MCP-1 secretion from monocytes on PEG-only hydrogels was Hck independent in contrast to Hck-dependent MCP-1 secretion in U937 on TCPS. Overall, U937 adherent to sIPNs secrete low levels of soluble gelatinase MMP-9, IL-1beta, TNF-alpha, IL-6, and MCP-1 independent of Hck and PAI-2. This work demonstrates significant changes in surface dependent expression of proteins from monocytes adherent to PEG-based materials compared to TCPS.

Figure 1

MMP-9 ELISA results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated with 50 ng/ml PMA and DMSO vehicle control, 10 μM PP2, or 10 μM PP3. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each). (■) = vehicle control; (▨) = PP2 in DMSO; (▯) = PP3 in DMSO. PMA⁻ (●) = 2053 ± 2052 pg/ml; LPS (▲) = 32,176 ± 4267 pg/ml. Supernatant from LPS-treated and U937 on TCPS were above detection limits.

Figure 2

TNF-α Bio-Plex results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated as in Figure 2. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each). (■) = vehicle control; (▨) = PP2; (▯) = PP3. * Different than TCPS within treatment group (p < 0.07). PMA⁻ (●) = 90.5 ± 264 pg/ml; LPS = 7683 ± 6422 pg/ml.

Figure 3

MCP-1 Bio-Plex results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated as in Figure 2. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each). (■) = vehicle control; (▨) = PP2; (▯) = PP3. * Different than TCPS within treatment group (p < 0.001); † Different than VC within surface (p ≤ 0.026); ‡ Different than PEG within treatment group (p ≤ 0.02). p = 0.053 for PP2 treatment on TCPS. PMA⁻ (●) = 409 ± 236 pg/ml; LPS = 229,823 ± 173,895 pg/ml.

Figure 4

MMP-9 ELISA results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated with 50 ng/ml PMA as the negative control or 50 ng/ml PMA with 10 IU uPA. U937 on TCPS and PEG hydrogels were also treated with 50 ng/ml PMA and 25 IU uPA during the second and third independent replicates. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each) except for 25 IU uPA (n = 2 independent replicates of 3 replicates each). (■) = negative control (i.e. 50 ng/ml PMA only); (▨) = 10 IU uPA; (▯) = 25 IU uPA. PMA⁻ (●) = 1835 ± 2443 pg/ml; LPS (▲) = 32,176 ± 4267 pg/ml. N/A = Not Analyzed.

Figure 5

PAI-2 ELISA results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated as in Figure 5. U937 on TCPS and PEG hydrogels were also treated with 50 ng/ml PMA and 25 IU uPA during the second and third independent replicates. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each) except for 25 IU uPA (n = 2 independent replicates of 3 replicates each). (■) = negative control (i.e. 50 ng/ml PMA only); (▨) = 10 IU uPA; (▯) = 25 IU uPA. PMA⁻ (●) = 18.0 ± 28.6 ng/ml; LPS (▲) = 149.9 ± 67.5 ng/ml. N/A = Not Analyzed. * Different than TCPS within treatment group (p < 0.05); ‡ Different than PEG within treatment group (p ≤ 0.034).

Figure 6

uPA ELISA results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated as in Figure 5. U937 on TCPS and PEG hydrogels were also treated with 50 ng/ml PMA and 25 IU uPA during the second and third independent replicates. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each) except for 25 IU uPA (n = 2 independent replicates of 3 replicates each). (■) = negative control (i.e. 50 ng/ml PMA only); (▨) = 10 IU uPA; (▯) = 25 IU uPA. PMA⁻ = −0.22 ± 0.16 ng/ml; LPS = −0.19 ± 0.21 ng/ml. N/A = Not Analyzed. * Different than TCPS within treatment group (p < 0.05); † Different than NC within surface (p ≤ 0.001); ‡ Different than PEG within treatment group (p ≤ 0.034); ◆ Different than 10 IU uPA (p ≤ 0.001).

Figure 7

uPAR ELISA results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated as in Figure 5. U937 on TCPS and PEG hydrogels were also treated with 50 ng/ml PMA and 25 IU uPA during the second and third independent replicates. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each) except for 25 IU uPA (n = 2 independent replicates of 3 replicates each). (■) = negative control (i.e. 50 ng/ml PMA only); (▨) = 10 IU uPA; (▯) = 25 IU uPA. PMA⁻ (●) = 203 ± 84 pg/ml; LPS (▲) = 1991 ± 795 pg/ml. N/A = Not Analyzed. * Different than TCPS within treatment group (p < 0.001); † Different than NC within surface (p ≤ 0.026); ‡ Different than PEG within treatment group (p ≤ 0.001).

Figure 8

TNF-α Bio-Plex results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated as in Figure 5. U937 on TCPS and PEG hydrogels were also treated with 50 ng/ml PMA and 25 IU uPA during the second and third independent replicates. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each) except for 25 IU uPA (n = 2 independent replicates of 3 replicates each). (■) = negative control (i.e. 50 ng/ml PMA only); (▨) = 10 IU uPA; (▯) = 25 IU uPA. PMA⁻ (●) = 2.11 ± 1.30 pg/ml; LPS (▲) = 6143 ± 3380 pg/ml. N/A = Not Analyzed. ‡ Different than PEG within treatment group (p ≤ 0.001).

Figure 9

MCP-1 Bio-Plex results on supernatant from U937 on TCPS, unmodified sIPN, RGD-modified sIPN, or PEG hydrogels at 24 h treated as in Figure 5. U937 on TCPS and PEG hydrogels were also treated with 50 ng/ml PMA and 25 IU uPA during the second and third independent replicates. Supernatant was tested in triplicate from three independent replicates (n = 3 independent replicates of 3 replicates each) except for 25 IU uPA (n = 2 independent replicates of 3 replicates each). (■) = negative control (i.e. 50 ng/ml PMA only); (▨) = 10 IU uPA; (▯) = 25 IU uPA. * Different than TCPS within treatment group (p ≤ 0.005); † Different than NC within surface (p ≤ 0.002); PMA⁻ (●) = 803 ± 570 pg/ml; LPS = 193,844 ± 237,097 pg/ml. N/A = Not analyzed. ‡ Different than PEG within treatment group (p ≤ 0.001); ◆ Different than 10 IU uPA (p ≤ 0.025).