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Polymodal Transient Receptor Potential Vanilloid (TRPV) Ion Channels in Chondrogenic Cells.

Somogyi CS, Matta C, Foldvari Z, Juhász T, Katona É, Takács ÁR, Hajdú T, Dobrosi N, Gergely P, Zákány R - Int J Mol Sci (2015)

Bottom Line: The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation.Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation.As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen H-4032, Hungary. somogyics@anat.med.unideb.hu.

ABSTRACT
Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

No MeSH data available.


mRNA expression of TRPVs in chicken (ch) and mouse (m) HD cultures. GAPDH was applied as an internal control. Numbers below gel images represent integrated densities of signals determined using ImageJ 1.46; data were normalised to the value detectable on the earliest day of culturing, i.e., day 0 (1.0) where applicable. Chicken TRPV5 expression was not analysed due to unpublished sequence data. GAPDH was used as an internal control. Representative data of three independent experiments.
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ijms-16-18412-f002: mRNA expression of TRPVs in chicken (ch) and mouse (m) HD cultures. GAPDH was applied as an internal control. Numbers below gel images represent integrated densities of signals determined using ImageJ 1.46; data were normalised to the value detectable on the earliest day of culturing, i.e., day 0 (1.0) where applicable. Chicken TRPV5 expression was not analysed due to unpublished sequence data. GAPDH was used as an internal control. Representative data of three independent experiments.

Mentions: In the following set of experiments, we screened the mRNA expression of various TRPVs in high density micromass cell cultures (HD). The advantage of the avian model over the murine model is that it is cost effective and yields higher initial cell number for the experiments. On the other hand, the amino acid sequences of TRPV proteins are different from the mammalian TRPV orthologues resulting in different responses to pharmacological modulations. Therefore, we decided to compare the mRNA expression of chicken and mouse HD cultures during the seven-day-culturing (Figure 2). The mRNA expression pattern of TRPV4 was the most distinct as it exhibited a steady elevation in both models. TRPV2 showed a relatively constant expression in both chicken and mouse HD cultures. In both experimental models TRPV1 and TRPV3 displayed a weaker signal towards the end of the culturing period. This was consistent in three independent chicken and mouse culture series and also confirmed by other TRPV1 and TRPV3 primers that were designed to amplify different sequences. Chicken TRPV6 primer pairs recognised two transcript variants with two different product lengths (348 bp for XM_004938143.1 and 447 bp for XM_004938142.1). Murine HD cultures displayed both TRPV5 and TRPV6 mRNAs whose expression pattern gradually decreased by the end of the culturing period. (The chicken and mouse TRPV GenBank IDs and the sequences of designed primers are listed in Table 1 and Table 2.)


Polymodal Transient Receptor Potential Vanilloid (TRPV) Ion Channels in Chondrogenic Cells.

Somogyi CS, Matta C, Foldvari Z, Juhász T, Katona É, Takács ÁR, Hajdú T, Dobrosi N, Gergely P, Zákány R - Int J Mol Sci (2015)

mRNA expression of TRPVs in chicken (ch) and mouse (m) HD cultures. GAPDH was applied as an internal control. Numbers below gel images represent integrated densities of signals determined using ImageJ 1.46; data were normalised to the value detectable on the earliest day of culturing, i.e., day 0 (1.0) where applicable. Chicken TRPV5 expression was not analysed due to unpublished sequence data. GAPDH was used as an internal control. Representative data of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581253&req=5

ijms-16-18412-f002: mRNA expression of TRPVs in chicken (ch) and mouse (m) HD cultures. GAPDH was applied as an internal control. Numbers below gel images represent integrated densities of signals determined using ImageJ 1.46; data were normalised to the value detectable on the earliest day of culturing, i.e., day 0 (1.0) where applicable. Chicken TRPV5 expression was not analysed due to unpublished sequence data. GAPDH was used as an internal control. Representative data of three independent experiments.
Mentions: In the following set of experiments, we screened the mRNA expression of various TRPVs in high density micromass cell cultures (HD). The advantage of the avian model over the murine model is that it is cost effective and yields higher initial cell number for the experiments. On the other hand, the amino acid sequences of TRPV proteins are different from the mammalian TRPV orthologues resulting in different responses to pharmacological modulations. Therefore, we decided to compare the mRNA expression of chicken and mouse HD cultures during the seven-day-culturing (Figure 2). The mRNA expression pattern of TRPV4 was the most distinct as it exhibited a steady elevation in both models. TRPV2 showed a relatively constant expression in both chicken and mouse HD cultures. In both experimental models TRPV1 and TRPV3 displayed a weaker signal towards the end of the culturing period. This was consistent in three independent chicken and mouse culture series and also confirmed by other TRPV1 and TRPV3 primers that were designed to amplify different sequences. Chicken TRPV6 primer pairs recognised two transcript variants with two different product lengths (348 bp for XM_004938143.1 and 447 bp for XM_004938142.1). Murine HD cultures displayed both TRPV5 and TRPV6 mRNAs whose expression pattern gradually decreased by the end of the culturing period. (The chicken and mouse TRPV GenBank IDs and the sequences of designed primers are listed in Table 1 and Table 2.)

Bottom Line: The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation.Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation.As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen H-4032, Hungary. somogyics@anat.med.unideb.hu.

ABSTRACT
Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

No MeSH data available.