Chondrocyte responses to neurovascular peptides, cytokines, and a 3D environment: focus on ADAMs

Figures & data

Table 1 Inventorized primer set codes for TLDA analysis and for further experimentation (ADAM28)

Gene	ABI inventorized primer set
ADAM9	Hs00177638_m1
ADAM10	Hs00153853_m1
ADAM12	Hs01106101_m1
ADAM15	Hs00984794_m1
ADAM17	Hs01041915_m1
ADAM19	Hs00224960_m1
ADAM20	Hs01083178s1
ADAM21	Hs01652548_s1
ADAM22	Hs00244640_m1
ADAM28	Hs00248020_m1
ADAM33	Hs00905552_m1
TIMP1	Hs00171558_m1
TIMP2	Hs00234278_m1
TIMP3	Hs00165949_m1
TIMP4	Hs00162784_m1
IL6	Hs00985639_m1
IL10	Hs00961622_m1
BDNF	Hs00380947_m1
NGF	Hs00171458_m1
NPY	Hs00173470_m1
Tachykinin 3	Hs00203109_m1
BDKRB1	Hs00664201_s1
BDKRB2	Hs00176121_m1
ADAM28	Forward: 5′- GGGCCCACGATTTGCA-3′ Reverse:
	5′-TGAACCTTCCTGTCTTTCAAIIIIACT-3′ Probe: 5′FAM-
	AGAACATTGCCCTACCTGCCACCAAAC-
	TAMRA-3

Abbreviations: ADAM, a disintegrin and metalloproteinase; TLDA, TaqMan low-density array; TIMP, tissue inhibitors of metalloproteinase; IL, interleukin; BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; NPY, neuropeptide Y; BDKRB, bradykinin receptor B.

Figure 1 Culture in 3D in general suppresses ADAM steady-state mRNA levels, irrespective of the stimulus added.

Notes: C28I/2 chondrocytes were cultured either in 2D (green) or 3D (red) with the indicated stimuli, and following RNA extraction and reverse transcription, TLDA analysis was performed. Each bar represents the mean ± SEM for triplicate samples following normalization to 18S RNA. For this fold-change analysis, control (unstimulated) cells were set as 1. Comparisons between the levels in 2D and 3D for control conditions and for each stimulus are indicated. *P<0.05; **P<0.02; ***P<0.01.

Abbreviations: 2D, two dimensional; 3D, three dimensional; TLDA, TaqMan low-density array; SEM, standard error of mean; ADAM, a disintegrin and metalloproteinase; IL, interleukin; OSM, oncostatin M.

Table 2 Modulation of steady-state mRNA levels for ADAMs, TIMPs, cytokines, and neurovascular factors under the indicated conditions, determined by TLDA analysis

Gene	3D versus 2D	ILI/OSM (2D)	IL1/OSM (3D)	SubP (2D)	SubP (3D)	BK (2D)	BK (3D)
ADAM9	−2.1	↔	−2.5	↔	−1.5	↔	−1.7
ADAM10	−1.7	2	↔	↔	↔	↔	↔
ADAM12	−1.7	4	2	↔	−1.7	↔	−1.7
ADAM15	−1.6	1.4	↔	↔	−1.4		−1.4
ADAM17	−1.4	↔	↔	1.4	↔	↔	↔
ADAM19	↔	1.7	−1.4	↔	−1.7	↔	↔
ADAM20	−2.7	2	↔	↔	↔	↔	↔
ADAM21	↔	↔	−2	↔	↔	↔	↔
ADAM22	↔	↔	↔	↔	−1.9	↔	↔
ADAM28	↔	↔	(2; P=0.09)	↔	↔	↔	↔
ADAM33	−2	↔	↔	↔	↔	↔	↔
TIMP1	↔	2	2.5	↔	↔	↔	↔
TIMP2	−1.5	↔	↔	↔	−1.5	↔	−1.7
TIMP3	↔	−2	−4	↔	−1.6	↔	↔
TIMP4	↔	↔	↔	↔	↔	↔	−1.8
IL6	−2	77	55	2.7	↔	↔	↔
IL10	5.9	65	13	↔	↔	↔	↔
BDNF	−2.3	−2.2	−3.5	↔	↔	↔	↔
NGF	−4	↔	↔	↔	↔	↔	↔
NPY	↔	−4.2	−3.5	↔	−1.7	↔	↔
Tachykinin 3	↔	−2.5	−2.1	↔	↔	↔	↔
BDKRB1	↔	5.3	4.1	1.7	↔	↔	↔
BDKRB2	↔	2.2	2	↔	↔	↔	↔

Notes: Significant fold changes are indicated compared to unstimulated cells in either 2D or 3D culture (or comparison between 2D and 3D for unstimulated cells only). ↔ indicates no change.

Abbreviations: IL1/OSM, interleukin 1/oncostatin M; subP, substance P; BK, bradykinin; ADAM, a disintegrin and metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; IL6, interleukin 6; IL10, interleukin 10; BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; NPY, neuropeptide Y; BDKRB1, bradykinin receptor B1; BDKRB2, bradykinin receptor B2; TLDA, TaqMan low-density array.

Figure 2 Confirmation of co-stimulatory effects of bradykinin and cytokines in chondrocytes cultured in 3D.

Notes: (A) ADAM28 steady-state mRNA levels are elevated by a combination of cytokines and bradykinin under normoxic conditions (red bars) and are maintained under hypoxic conditions (blue). (B) IL6 expression is synergistically enhanced by the combination of cytokines and bradykinin under normoxic conditions (red) and overall responses are reduced under hypoxic conditions (blue). Steady-state mRNA levels for ADAM28 and IL6 were determined by qRT-PCR. For this fold-change analysis, control (unstimulated) cells were set as 1. Each bar represents the mean ± SEM for triplicate samples following normalization to 18S RNA. *P<0.05; **P<0.02; ***P<0.01.

Abbreviations: ADAM, a disintegrin and metalloproteinase; IL, interleukin; OSM, oncostatin M