Objective
Infection triggers inflammation that, in turn, enhances the expression of contractile-associated factors in myometrium and increases the risk of preterm delivery. In this study, we assessed vitamin D regulation of inflammatory markers, contractile-associated factors, steroid hormone receptors, and NFκB pathway proteins in human uterine myometrial smooth muscle (UtSM) cells that were cultured in an inflammatory environment.
Study Design
Inflammatory environment was simulated for UtSM cells by coculturing them with monocyte lineage (THP1) cells. We measured the expression of inflammatory markers, contractile-associated factors, steroid hormone receptors, and NFκB pathway proteins in UtSM cells that were cultured with THP1 cells in the presence and absence of vitamin D by real time polymerase chain reaction and Western blot analysis.
Results
Monocytes secreted monocyte inflammatory protein–1α and –1β, interleukin (IL)-1β and 6, and tumor necrosis factor–α into the conditioned medium. In the UtSM cells that had been cocultured with THP1 cells, there was a significant ( P < .05) increase in the expression of inflammatory markers IL-1β, -6, and -13 and tumor necrosis factor–α; the contractile-associated factors connexin-43, Cox-2, and prostaglandin F 2 α receptor; the estrogen receptor α, and progesterone receptors A and B. Vitamin D treatment of cocultures decreased ( P < .05) the expression of inflammatory markers and contractile-associated factors in UtSM cells. Similarly, vitamin D decreased estrogen receptor α and progesterone receptors A-to-B ratio in UtSM cells that were cocultured with THP1 cells. In addition, vitamin D treatment significantly ( P < .05) decreased monocyte-induced p-IκBα in cytosol and NFκB-p65 in the nucleus and increased IκBα in cytosol in UtSM cells.
Conclusion
Our results suggest that vitamin D treatment decreases inflammation-induced cytokines and contractile-associated factors in the uterine myometrial smooth muscle cells through the NFκB pathway.
Infection induces inflammatory reactions mainly through the production of cytokines. Inflammation is one of the responses of the body to infection, to remove the injurious stimuli, and to initiate the healing process. In addition, controlled aseptic inflammations were reported to play a role in normal parturition at term. On the other hand, untimely activation of inflammation has been implicated in preterm labor. Infection not only initiates inflammatory responses but also was reported to be responsible for 40% of the premature births. Cell-derived and plasma-derived mediators play a role in infection-induced inflammatory reactions. Lipopolysaccharide, a bacterial endotoxin has been reported to increase the cytokine secretion in human myometrial cells, enhance prostaglandin production in rodent and human myometrium and cause preterm delivery in rodents. In turn, cytokine treatments have been reported to increase prostaglandins, connexin-43, oxytocin, and oxytocin receptor, which suggests that infection increases cytokines that, in turn, increase contractile-associated proteins and initiate preterm delivery.
The expression levels of progesterone receptor A and estrogen receptor α were reported to be high not only at term but also in preterm deliveries. Even though infection was reported to increase inflammation and cause preterm deliveries, it is not clear whether the expression of the progesterone and the estrogen receptors was altered in myometrium in infection-induced inflammation. Furthermore, infection was reported to induce phosphorylation of IκBα, which leads to the translocation of NFκB into nucleus, increase the production of the proinflammatory cytokines, and increase the expression of contractile-associated factors. Vitamin D was reported to play a role in antagonizing the inflammatory effect of infections in addition to its classic effect on calcium and phosphate homeostasis. Studies showed that vitamin D treatment has been used successfully to treat tuberculosis. In addition, recent findings suggest that vitamin D improves the cytokine profile in patients with congestive heart failure and inhibits both cytokines and contractile-associated proteins in myometrial cells that are treated with lipopolysaccharide. Vitamin D has also been reported to regulate NFκB pathway proteins in decidual cells. However, the effect of vitamin D action on myometrial cells in a biologic system that mimics in vivo conditions and examines cross-talk between immune and nonimmune cells within the uterus is not yet defined. In this study, we used monocyte lineage (THP1) cells to simulate an in vivo environment of inflammation for uterine smooth muscle cells and assess whether vitamin D regulates proinflammatory cytokines, contractile-associated proteins, steroid hormone receptor expression, and NFκB pathway proteins in human uterine myometrial smooth muscle (UtSM) cells.
Materials and Methods
Cell lines and reagents
The UtSM cell line was a generous gift from Dr Darlene Dixon (National Institute of Environmental Health Sciences). THP1 cells were obtained from ATCC (Manassas, VA). Smooth muscle basal medium, growth factors, antibiotics, gentamicin sulphate, and amphotericin B were obtained from Lonza Walkersville Inc (Walkersville, MD). Roswell Park Memorial Institute (RPMI) medium and connexin-43 antibody were obtained from Life Technologies (Grand Island, NY). Vitamin D3, absolute alcohol, and β-actin antibody were obtained from Sigma-Aldrich Co (St. Louis, MO). SYBR green was obtained from Bio-Rad Laboratories (Hercules, CA). Progesterone receptor and estrogen receptor α antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Cox-2 antibody from Cayman Chemicals (Ann Arbor, MI), and NFκB-p65, IκBα, and p-IκBα antibodies from Abcam Inc (Cambridge, MA).
Cell culture
Immortalized adherent UtSM cells were cultured in smooth muscle basal medium supplemented with 5% fetal bovine serum, human epidermal growth factor (0.1%), recombinant human fibroblast growth factor (0.2%), 0.1% insulin, and 0.1% antibiotics gentamicin sulphate and amphotericin B. These cells were maintained at 37°C in a humidified atmosphere of air and 5% CO 2 .
Monocyte culture
Nonadherent THP1 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 2 mmol/L glutamine, 1 mmol/L HEPES, and 0.05 mmol/L β-mercaptoethanol (Sigma-Aldrich Co). They were maintained at 37°C in a humidified atmosphere of air and 5% CO 2 .
Coculture
UtSM cells that had been grown to 90% confluence in 60-mm dishes were starved for 24 hours in RPMI medium that contained 0.1% bovine serum albumin (Sigma-Aldrich Co), sodium pyruvate, glutamine, HEPES, and β-mercaptoethanol. Inflammatory environment was created for UtSM cells by coculturing them with THP1 (0.2×10 6 ) cells. Cocultures of UtSM and THP1 cells were treated in triplicates with 0 (control), 5, 10, 50, 150, and 300 nmol/L of 1,25 (OH) 2 vitamin D (Sigma Chemical Co) dissolved in alcohol (Sigma Chemical Co). Myometrial cells cultured alone served as normal control. Cocultures were maintained at 37°C in a humidified atmosphere of air and 5% CO 2 on a platform shaker at slow speed. After the treatment, adherent UtSM cells were washed once with diluted Trypsin-EDTA (Life Technologies) and twice with ice cold phosphate-buffer saline solution to remove monocytes that might have been attached to the adherent myometrial cells.
Chemokine and cytokine assay
Conditioned media were collected from monocyte (0.2×10 6 ) and UtSM cells that had been cultured separately. Monocytes in conditioned medium were removed by centrifugation at 10,000 rpm at 4°C, and the supernatant was stored at –70°C until further analysis.
We measured the levels of monocyte inflammatory protein (MIP)–1α and –1β, interleukin (IL)-1β and -6, and tumor necrosis factor alpha (TNFα) in the conditioned media that were obtained from monocytes and UtSM cell cultures by multiplex assay system (Luminex 100 system; BioRad, Austin, TX) in picograms/milliliter (pg/mL).
Isolation of total RNA and reverse transcription
Total RNA was extracted with an RNeasy kit (Qiagen-Xeragon, Valencia, CA) from the UtSM cells that had been cultured alone and from UtSM cells that had been cocultured with THP1 cells in the presence and absence of vitamin D. RNA was treated with DNAse I (Qiagen-Xeragon) enzyme to remove genomic DNA contamination. As described elsewhere, 1.0 μg of total RNA was briefly mixed with 3.0 nmol of oligo dT, 200 μmol/L dNTP, 5 U RNase inhibitor, and 10 U of avian myeloblastosis virus reverse transcriptase (Promega Corp, Madison, WI) in a total volume of 20 μL. Complementary DNA was generated by reverse transcription in a thermal cycler set at 70°C for 5 minutes, 25°C for 10 minutes, and 42°C for 60 minutes and was stored at –20°C until analysis.
Real time polymerase chain reaction
Briefly, 50 ng of complementary DNA was added to the master mix that contained SYBR green (Bio-Rad Laboratories) and specific primers for IL-1β, -6, and -13, TNFα, connexin-43, prostaglandin receptor, oxytocin receptor, and glyceraldehyde 3-phosphate dehydrogenase. Primer sequences were obtained from peer reviewed publications ( Table 1 ). All primer sets that were used for real time polymerase chain reaction (RT-PCR) generated a single amplicon. RT-PCR was performed with a Bio-Rad MyiQ5. Genes were amplified at 95°C for 15 seconds and 60°C for 1 minute for 40 cycles. The resulting data were normalized with respective GAPDH values.
| Gene | Primer sequence | Amplicon length | NCBI Accession # |
|---|---|---|---|
| IL-1β Forward | 5′-CAA ATT CGG TAC ATC CTC GAC-3′ | 73 | NM_000576 |
| IL-1β Reverse | 5′-GTC AGG GGT GGT TAT TGC ATC-3′ | ||
| IL6 Forward | 5′-AAA CAG ATG AAG TGC TCC TTC CAG G-3′ | 339 | NM_000600 |
| IL6 Reverse | 5′-TGG AGA ACA CCA CTT GTT GCT CCA-3′ | ||
| IL13 Forward | 5′-TGA GGA GCT GGT CAA CAT CA-3′ | 76 | NM_002188 |
| IL13 Reverse | 5′-CAG GTT GAT GCT CCA TAC CAT-3′ | ||
| TNFα Forward | 5′-CAG AGG GAA GAG TTC CCC AG-3′ | 84 | NM_000594 |
| TNFα Reverse | 5′-CCT TGG TCT GGT AGG AGA CG-3′ | ||
| Connexin-43 Forward | 5′-CCT ATG TCT CCT CCT GGG TA-3′ | 176 | NM_000615 |
| Connexin-43 Reverse | 5′-GGG AAA TCA AAA GGC TGT G-3′ | ||
| Prostaglandin F2α Receptor Forward | 5′-GCA GCT GCG CTT CTT TCA A-3′ | 81 | NM_000959 |
| Prostaglandin F2α Receptor Reverse | 5′-CAC TGT CAT GAAGAT TAC TGA AAA AAA TAC-3′ | ||
| Oxytocin Receptor Forward | 5′-CTG AAC ATC CCG AGG AAC TG-3′ | 84 | NM_000916 |
| Otyocin Receptor Reverse | 5′-CTC TGA GCC ACT GCA AAT GA-3′ | ||
| GAPDH Forward | 5′-TGA TGA CAT CAA GAA GGT GGT-3′ | 240 | NM_002046 |
| GAPDH Reverse | 5′-TCC TTG GAG GCC ATG TGG GCC-3′ |
Whole cell lysates and cytosol preparation
Whole cell lysates, cytosol, and nuclear fractions were prepared from UtSM cells as described by Suzuki et al with slight modifications. Briefly, monolayer cells in 60-mm dishes were washed once with diluted Trypsin-EDTA and twice with ice cold phosphate-buffered saline solution to remove any attached monocytes. Lysates were prepared using radioimmunoprecipitation assay buffer that contained 1X protease inhibitor cocktail, 1 mmol/L sodium vanadate, 0.1% NP40, and 1 mmol/L sodium fluoride (Sigma Chemical Co). Part of the whole cell lysate was subjected to brief sonication with a microprobe from Misonix Incorporated (Farmingdale, NY) and stored at –80°C until analysis. Remaining lysate was centrifuged at 10,000 rpm for 10 minutes at 4°C on a bench top centrifuge. Supernatant (cytosol) was stored at –80°C until further analysis. The pellet was used for nuclear protein preparation.
Nuclear protein preparation
Pelleted nuclei were suspended in 75 μL of radioimmunoprecipitation assay buffer that contained 1X protease inhibitor cocktail and 1 mmol/L dithiothreitol (Sigma Chemical Co) and incubated for 15 minutes on ice. Nuclear pellets were sonicated twice for 5 seconds each with microprobes from Misonix Incorporated and centrifuged at 16000 g for 15 minutes at 4°C. Supernatants that contained the nuclear proteins were stored at –80°C until analysis.
Western blot analysis
Equal amounts of protein were resolved on a 10% Tris-Bis gels and transferred onto polyvinylidene difluoride membranes. Western blot analysis was performed with primary antibodies against Cox-2 (1:500), connexin-43 (1:500), estrogen receptor α (1:500), progesterone receptors A (1:500) and B (1:250), IκBα, p-IκBα, NFκB-p65 (2 ng/mL) and β-actin (1:5000). The protein signal intensity was quantified with an image documentation system (ProteinSimple, Santa Clara, CA) and normalized with the corresponding β-actin values.
Results
Monocytes secreted chemokines and cytokines into culture media
We measured chemokines and cytokines in conditioned media that were obtained from both monocytes and UtSM cells. Chemokines MIP-1α and -1β, cytokines IL-1β and -6, and TNFα were significantly higher in monocyte cultures, although their levels were below the detection limit in UtSM cultures ( Table 2 ).
| Cells | Monocyte inflammatory protein–1α, pg/mL | Monocyte inflammatory protein–1β, pg/mL | Interleukin-1β, pg/mL | Tumor necrosis factor α, pg/mL | Interleukin-6, pg/mL |
|---|---|---|---|---|---|
| Human uterine myometrial smooth muscle | <3.20 | <3.20 | <3.20 | <3.20 | <3.20 |
| THP1 cells | 86.61 ± 11 | 245.00 ± 45 | 5.20 ± 1.1 | 8.48 ± 1.5 | 10.06 ± 2.0 |
Vitamin D decreased messenger RNA (mRNA) expression of proinflammatory markers in UtSM cells that had been cocultured with monocytes
RT-PCR was performed to assess the effects of vitamin D on inflammatory cytokines in UtSM cells that had been cocultured with monocytes. UtSM cells when cultured with THP1 cells showed a significant increase ( P < .01) in the mRNA expression of IL-1β, -6, and -13 and TNFα. However, treatment of cocultures with vitamin D significantly decreased ( P < .05) their expression in UtSM cells when compared with that in the vehicle treated cocultures ( Figure 1 ).
Vitamin D reduced monocyte-induced contractile-associated proteins in uterine myometrial smooth muscle cells
We measured the mRNA expression of contractile-associated proteins in UtSM cells that had been cocultured with monocytes in the presence and absence of vitamin D. RT-PCR analysis showed a significant increase in the expression of connexin-43, prostaglandin F2α receptor, and oxytocin receptor in UtSM cells that had been cocultured with monocytes ( P < .05; Figure 2 ). However, treatment of cocultures with vitamin D significantly decreased their expression in UtSM cells in comparison with that in the vehicle treated cocultures ( P < .05; Figure 2 ).
Western blot analysis showed similar increases in Cox-2 and connexin-43 expression in UtSM cells that had been cocultured with monocytes ( P < .05); vitamin D treatment significantly decreased their expression in comparison with that in the vehicle treated cocultures ( P < .05; Figure 3 ).
Vitamin D decreased estrogen receptor α in UtSM cells that had been cocultured with monocytes
We measured estrogen receptor α expression in UtSM cells that had been cocultured with monocytes and treated with various concentrations of vitamin D. Western blot analysis of nuclear protein that was obtained from UtSM cells that had been cocultured with monocytes showed a significant increase in the expression of estrogen receptor α in UtSM cells in coculture when compared with vehicle-treated control ( P < .01; Figure 4 ). Vitamin D treatment of cocultures significantly decreased estrogen receptor α expression in UtSM cells at 150 and 300 nmol/L concentrations when compared with that in the vehicle-treated cocultures ( P < .01).
