Action of 1,25(OH)₂D₃ on Human Asthmatic Bronchial Fibroblasts: Implications for Airway Remodeling in Asthma

Figures & data

Table 1 Primers Used for Semi-Quantitative RT-PCR

Gene	Forward Primer Sequence (5ʹ to 3ʹ)	Reverse Primer Sequence (5ʹ to 3ʹ)	Product Size (bp)
GAPDH	GAAGGTGAAGGTCGGAGT	GAAGATGGTGATGGGATTTC	226
VDR	CTTCAGGCGAAGCATGAAGC	CCACCATCATTCACACGAACTGG	128
CYP24A1	GCTTCTCCAGAAGAATGCAGGG	CAGACCTTGGTGTTGAGGCTCT	125
FN 1	CCAACTGGTAACCCTTCC	CCAACACTGGGTTGCTGA	156
LUM	AACATACCAACTGTCAATGAAAACC	TGCCATCCAAACGCAAATGCTTG	125
BGN	TTGAACCTGGAGCCTTCGATGG	TTGGAGTAGCGAAGCAGGTCCT	150
MMP2	AGCGAGTGGATGCCGCCTTTAA	CATTCCAGGCATCTGCGATGAG	137
TIMP1	GGAGAGTGTCTGCGGATACTTC	GCAGGTAGTGATGTGCAAGAGTC	100
COL5A1	GGAGATGATGGTCCCAAAGGCA	CCATCATCTCCTTTGTCACCAGG	118
CCL2	CCCCAGTCACCTGCTGTTAT	TGGAATCCTGAACCCACTTC	171
CCL5	CGCTGTCATCCTCATTGCTA	GAGCACTTGCCACTGGTGTA	147
CCL11	AATGTCCCCAGAAAGCTGTG	TCAGGCTCTGGTTTGGTTTC	163
CCND1	CAATGACCCCGCACGATTTC	AAGTTGTTGGGGCTCCTCAG	208
BCL2	CTTTGAGTTCGGTGGGGTCA	GGGCCGTACAGTTCCACAAA	162
BAX	TCAGGATGCGTCCACCAAGAAG	TGTGTCCACGGCGGCAATCATC	103

Figure 1 mRNA expression of VDR (A) and CYP24A1 (B) in NHBFCs or DHBFCs as a response to 1,25(OH)₂D₃ (50 nM or 100 nM) treatment. Student t-test was applied to compare the fold change difference in mRNA expression between two experimental conditions and associated p- values are indicated. (ns) p > 0.05, no significant difference in mean fold change mRNA expression. Data were expressed as mean ± standard error (SE) from duplicate values of two independent experiments. NHBFCs (n=4), DHBFCs (n=4).

Figure 2 mRNA expression of TGF-β1 or TNF-α-IL-1β-induced fibrogenic markers/CCL-chemokines in NHBFCs or DHBFCs in response to 1,25(OH)₂D₃ (50 nM) treatment: FN 1 (A), LUM (B), BGN (C), COL5A1 (D), MMP2 (E), TIMP1 (F), CCL2 (G), CCL5 (H) and CCL11 (I). The Student’s t-test (variances assumed equally by ANOVA) was used to determine the difference between NHBFCs and DHBFCs under a given treatment and associated p- values are indicated. (ns) p > 0.05, no significant difference in mean fold change mRNA expression. Data were expressed as mean ± standard error (SE) from duplicate values of two independent experiments. NHBFCs (n=4), DHBFCs (n=4).

Table 2 The mRNA Expression and Protein Level of Fibrogenic Markers and CC-Chemokines, Intra-and Inter-Groups’ Comparisons

Inter-Groups’ NHBFCs vs DHBFCs Comparisons
Fibrogenic Markers	TGF-β1 or TNF-α-IL-1β-NHBFCs vs TGF-β1 or TNF-α-IL-1β-DHBFCs		1,25(OH)2D3-TGF-β1 or TNF-α-IL-1β-NHBFCs vs 1,25(OH)2D3-TGF-β1 or TNF-α-IL-1β-DHBFCs
	mRNA Expression (Mean Difference ± SEM)	p-values	mRNA Expression (Mean Difference ± SEM)	p-values
FN 1	3 ± 1.2	> 0.05	0.77 ± 0.3	> 0.05
LUM	0.35 ± 0.16	> 0.05	0.22 ± 0.17	> 0.05
BGN	4.425 ± 0.8	0.045	1.77 ± 0.4	0.008
COL5A1	0.88 ± 0.3	> 0.05	0.26 ± 0.19	> 0.05
MMP2	0.47 ± 0.07	> 0.05	0.05 ± 0.06	> 0.05
TIMP1	0.27± 0.1	> 0.05	2.9 ± 0.29	0.0009
CCL2	72.13 ± 19	0.03	20.98 ± 5.7	> 0.05
CCL5	519 ± 86	0.019	149.4 ± 25.64	0.025
CCL11	5.9 ± 2.3	0.04	3.1 ± 0.37	0.028
a	Protein levels (Mean difference ± SEM)	p- values	Protein levels (Mean difference ± SEM)	p- values
Fibronectin 1	330.2 ± 62.7	0.018	131 ± 36	0.02
Lumican	3693 ± 500.6	0.008	2140 ± 600	0.02
MCP1	163.9 ± 34.09	0.03	47 ± 27.2	> 0.05
RANTES	50.8 ± 65	> 0.05	1.632 ± 93	> 0.05
Eotaxin-1	101.2 ± 20	0.0065	76.16 ± 16	0.045
Intra-groups’ NHBFCs or DHBFCs vs unstimulated NHBFCs comparisons
Fibrogenic Markers	TGF-β1 or TNF-α-IL-1β-NHBFCs vs 1,25(OH)2D3-TGF-β1 or TNF-α-IL-1β-NHBFCs		TGF-β1 or TNF-α-IL-1β-DHBFCs vs 1,25(OH)2D3-TGF-β1 or TNF-α-IL-1β-DHBFCs
	mRNA expression (Mean difference ± SEM)	p- values	mRNA expression (Mean difference ± SEM)	p- values
FN 1	1.65 ± 0.25	0.045	3.4 ± 0.59	0.036
LUM	0.85 ± 0.15	0.0005	0.96 ± 0.14	0.0025
BGN	2.6 ± 0.25	0.0043	5.3 ± 0.45	0.0016
COL5A1	0.95 ± 0.18	0.001	1.6 ± 0.42	0.0086
MMP2	0.6 ± 0.05	0.02	0.93 ± 0.12	0.0025
TIMP1	3.4 ± 0.17	0.0001	0.66 ± 0.1	0.05
CCL2	29.1 ± 9.4	0.045	80.3 ± 5.7	0.001
CCL5	74 ± 12.3	0.001	494 ± 56	0.031
CCL11	1.05 ± 0.5	0.05	3.8 ± 1.37	0.04
	Protein levels (Mean difference ± SEM)	p- values	Protein levels (Mean difference ± SEM)	P- values
Fibronectin 1	51.82 ± 31.3	> 0.05	250 ± 55.3	0.03
Lumican	246.7 ± 89.9	> 0.05	1800 ± 607	0.045
MCP1	76.9 ± 60	> 0.05	193 ± 34	0.013
RANTES	209 ± 42	0.045	258 ± 75	0.04
Eotaxin-1	6.2 ± 5.1	> 0.05	62.31 ± 16	0.038

Notes: HBFCs were stimulated by TGF-β1 or TNF-α-IL-1β (10 ng/mL each cytokine), in the presence or absence of 50 nM of 1,25(OH)₂D₃. mRNA expression of fibrogenic markers/CC-chemokines in HBFCs was quantified by RT-qPCR, while protein quantification in HBFCs’ culture media was performed by ELISA. Italic p- values represent not significant (ns) difference, where p > 0.05.

Abbreviation: SEM, standard error of the mean.

Figure 3 Protein levels of TGF-β1 or TNF-α-IL-1β-induced fibrogenic markers/CCL-chemokines in NHBFCs or DHBFCs in response to 1,25(OH)₂D₃ (50 nM): fibronectin 1 (A), lumican (B), MCP1 (C), RANTES (D) and eotaxin-1 (E). The Student’s t-test (variances assumed equally by ANOVA) was used to determine the difference between NHBFCs and DHBFCs under a given treatment and associated p- values are indicated. (ns) p > 0.05, no significant difference. Data were expressed as mean ± standard error (SE) from duplicate values of two independent experiments. NHBFCs (n=4), DHBFCs (n=4).

Figure 4 Effect of 1,25(OH)₂D₃ (50 nM) on HBFCs proliferation using flow cytometry (A–G). HBFCs have been measured for 7AAD or BrdU content and were assigned to the G0/G1, S or G2/M phases by drawing gates, as demonstrated in each panel: TGF-β1-treated NHBFCs (A), 1,25(OH)₂D₃-TGF-β1-treated NHBFCs (B), TGF-β1-treated NHBFCs vs 1,25(OH)₂D₃-TGF-β1-treated NHBFCs, overlapped data (C), TGF-β1-treated DHBFCs (D), 1,25(OH)₂D₃-TGF-β1-treated DHBFCs (E), TGF-β1-treated DHBFCs vs 1,25(OH)₂D₃-TGF-β1-treated DHBFCs, overlapped data (F). The percentage of cells in each gate represents the relative number of NHBFCs or DHBFCs in G0/G1, S, and G2/M. (G) Graphic quantitation of the respective cell cycle phases using GraphPad. (H) mRNA expression of CCND1 in TGF-β1-induced-DHBFCs or NHBFCs in response to 1,25(OH)₂D₃ (50 nM) treatment. The Student’s t-test (variances assumed equally by ANOVA) was used to determine the difference between NHBFCs and DHBFCs under a given treatment and associated p- values are indicated. (ns) p > 0.05, no significant difference. Data were expressed as mean ± standard error (SE) from duplicate values of two independent experiments. NHBFCs (n=4), DHBFCs (n=4).

Figure 5 Effect of 1,25(OH)₂D₃ (50 nM) on HBFCs apoptosis using flow cytometry (A–G). HBFCs have been measured for Annexin V-FITC or 7AAD content and assigned to different cell populations by drawing gates, as demonstrated in each panel: Q1 (necrotic cells), Q2 (late apoptotic cells), Q3 (early apoptotic cells) and Q4 (living intact cells). TGF-β1-treated NHBFCs (A), 1,25(OH)₂D₃-TGF-β1-treated NHBFCs (B), TGF-β1-treated NHBFCs vs 1,25(OH)₂D₃-TGF-β1-treated NHBFCs, overlapped data (C), TGF-β1-treated DHBFCs (D), 1,25(OH)₂D₃-TGF-β1-treated DHBFCs (E), TGF-β1-treated DHBFCs vs 1,25(OH)₂D₃-TGF-β1-treated DHBFCs, overlapped data (F). The population percentage in each quadrant is also indicated. (G) Graphic quantitation of the respective cell populations using GraphPad. (H) mRNA expression of BAX in 1,25(OH)₂D₃ (50 nM) treated HBFCs. The Student’s t-test (variances assumed equally by ANOVA) was used to determine the difference between NHBFCs and DHBFCs under a given treatment and associated p- values are indicated. (ns) p > 0.05, no significant difference. Data were expressed as mean ± standard error (SE) from duplicate values of two independent experiments. NHBFCs (n=4), DHBFCs (n=4).