Research ArticleBONE DEVELOPMENT

TRPM7 channels mediate spontaneous Ca2+ fluctuations in growth plate chondrocytes that promote bone development

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Science Signaling  09 Apr 2019:
Vol. 12, Issue 576, eaaw4847
DOI: 10.1126/scisignal.aaw4847
  • Fig. 1 Growth plate chondrocytes exhibit spontaneous Ca2+fluctuations.

    (A) Representative fluorescence image of a Fluo-4–loaded femoral growth plate slice prepared from an E17.5 mouse. The zones of round (R), columnar (C), and hypertrophic (H) chondrocytes are indicated. Scale bar, 100 μm. (B) Fura-2 imaging of round and columnar chondrocytes under basal conditions. Representative images of recorded cells and Fura-2 ratiometric recordings derived from individual Ca2+ fluctuation–positive cells (red and green traces) and Ca2+ fluctuation–negative cells (blue and black traces) are shown. Data are representative of ≥19 cells from ≥5 mice. Scale bar, 20 μm. (C) Quantification of the fraction of round and columnar chondrocytes exhibiting Ca2+ fluctuations and the resting [Ca2+]i in fluctuation-negative and fluctuation-positive round and columnar chondrocytes. Ca2+ fluctuation–positive cells were defined as growth plate chondrocytes exhibiting at least one spontaneous event (>0.025 in Fura-2 ratio) during a 270-s observation window under basal conditions. Statistical analysis of resting [Ca2+]i revealed no difference between fluctuation-positive and fluctuation-negative populations in either round or columnar chondrocytes (t test). (D) Quantification of the amplitude, duration, and frequency of spontaneous Ca2+ events in fluctuation-positive round and columnar chondrocytes. The fura-2 ratio (F340/F380) was measured and calibrated for estimating [Ca2+]i as described in Materials and Methods. Data represent means ± SD. The numbers of cells and mice examined are shown in parentheses in the keys and above the graph bars, respectively.

  • Fig. 2 Ca2+influx is essential for Ca2+fluctuations in chondrocytes.

    (A) Fura-2 imaging of round chondrocytes in Ca2+-free bathing solution or in the presence of the calcium channel blocker Gd3+. Representative traces from individual fluctuation-positive cells are shown in the recordings, and the observations are summarized in the bar graph. (B) Fura-2 imaging of round chondrocytes in the presence of TG or CPA. Representative traces recorded from individual fluctuation-positive (red and green) and fluctuation-negative (black) cells are shown, and the observations are summarized in the bar graphs. Data represent means ± SEM, and the numbers of cells and mice examined are shown in parentheses in the keys and above the graph bars, respectively. Significant differences between values before and after the treatments are marked with asterisks [*P < 0.05 and **P < 0.01 in one-way analysis of variance (ANOVA) and Dunnett’s test]. DMSO, dimethyl sulfoxide.

  • Fig. 3 TRPM7 modulators alter Ca2+fluctuations in chondrocytes.

    (A) Fura-2 imaging of round chondrocytes in the presence of TRPM7 inhibitors (FTY720 and NS8593) or activators (naltriben and NNC550396). Representative traces recorded from individual round chondrocytes are shown. (B) Quantification of the effects of TRPM7 modulators on the fraction of cells that exhibited Ca2+ fluctuations and the amplitude of the fluctuations. Data represent means ± SEM, and the numbers of cells and mice examined are shown in parentheses in the keys and above the graph bars, respectively. Significant differences between the values before and after the modulator treatments are marked with asterisks (*P < 0.05 and **P < 0.01 in one-way ANOVA and Dunnett’s test).

  • Fig. 4 Trpm7 knockdown reduces Ca2+fluctuations in chondrocytes.

    (A) Representative Fura-2 recording traces from fluctuation-positive (red and green) and fluctuation-negative (blue and black) round chondrocytes from tamoxifen-treated Trpm7fl/fl (Cre-ER−/−) and Trpm7fl/fl (Cre-ER+/−) mice. The cells were examined under basal conditions and in the presence of the TRPM7 activator naltriben. (B) Quantification of the effects of Trpm7 knockdown on Ca2+ fluctuations under basal and naltriben-treated conditions. Data represent means ± SEM, and the numbers of cells and mice examined are shown in parentheses in the keys and above the graph bars, respectively. Significant differences between the Cre-ER−/− and Cre-ER+/− cells are marked with asterisks (*P < 0.05 and **P < 0.01 in t test). (C) Scatterplot to evaluate the correlation between Ca2+ fluctuation amplitudes under basal and naltriben-treated conditions in round chondrocytes from Trpm7 knockdown (filled circles) and control (open circles) embryos. Each circle represents one cell. In this diagram, the regression line based on all plots from both cell populations is drawn according to the linear least-squares method, and the Pearson R value was determined.

  • Fig. 5 PLC and BK channel modulators alter Ca2+fluctuations in chondrocytes.

    (A) Representative Fura-2 recording traces from fluctuation-positive and fluctuation-negative round chondrocytes in the presence of the PLC inhibitor U73122, the inactive analog U73343, the BK channel blocker paxilline, or the BK channel opener NS1619. (B) Quantification of the effects of U73122, U73343, paxilline, and NS1619 on Ca2+ fluctuations. Data represent means ± SEM, and the numbers of cells and mice examined are shown in parentheses in the keys and above the graph bars, respectively. Statistical differences between DMSO (vehicle control) and modulator treatments are marked with asterisks (*P < 0.05 and **P < 0.01 in one-way ANOVA and Dunnett’s test). (C) Proposed functional coupling of TRPM7 channel activity with PLC-mediated PI turnover and cellular K+ efflux through BK channels in growth plate chondrocytes. TRPM7-mediated Ca2+ entry seems to be essential for growth plate chondrogenesis to promote bone outgrowth. PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate.

  • Fig. 6 Defective outgrowth and chondrogenesis in cultured Trpm7-deficient bones.

    (A) Outgrowth of cultured Trpm7-deficient metatarsal bones. Representative images of metatarsals from Trpm7fl/fl (Cre-ER+/−) embryos before and after 8 days of culture in the presence or absence of 4OH-Tam are shown. Scale bar, 0.3 mm. In the right graph, longitudinal growth of metatarsals from Trpm7fl/fl (Cre-ER+/−) and control [Trpm7fl/fl (Cre-ER−/−)] embryos cultured in the presence or absence of 4OH-Tam was quantified over the culture period. The bone sizes on culture day 8 were statistically analyzed between groups. (B) Histological analysis of Trpm7-deficient metatarsal bones. Cross sections of bones from Trpm7fl/fl (Cre-ER+/−) mice cultured in the presence or absence of 4OH-Tam show the growth plate images and high-magnification views of the round (R), columnar (C), and hypertrophic (H) chondrocyte zones. Scale bars, 100 μm. (C) Summary of graphical representations of zonal areas containing round, columnar, and hypertrophic chondrocytes and their size of cells in each zone. Data represent means ± SEM, and the numbers of mice examined are shown in parentheses in the keys. Significant differences between the groups are marked with asterisks (*P < 0.05, **P < 0.01 in t test).

  • Fig. 7 Inactivated CaMKII signaling in cultured Trpm7-deficient bones.

    (A) Immunoblotting for and quantification of CaMKII, phosphorylated CaMKII (pCaMKII), CREB, and phosphorylated CREB (pCREB) in growth plates from cultured Trpm7-deficient bones. Growth plate extracts were prepared from Trpm7fl/fl (Cre-ER+/−) femoral bones cultured for 8 days in the presence or absence of 4OH-Tam and subjected to Western blot analysis and subsequent quantification by densitometry. The antibodies used for detecting total CaMKII and phosphorylated CaMKII react with all CaMKII subtypes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a loading control. Cultured Trpm7-deficient bones from six mice were analyzed. (B) Expression of the indicated transcripts in cultured Trpm7-deficient bones. Total RNAs were prepared from Trpm7fl/fl (Cre-ER+/−) metatarsal bones cultured for 8 days in the presence or absence of 4OH-Tam and subjected to RT-PCR analysis using the primer sets listed in table S1. The cycle threshold (Ct) value determined for each reaction was normalized to the internal control Gapdh value using the comparative Ct method. Data represent means ± SEM, and the numbers of mice examined are shown in parentheses. Significant differences between the groups are marked with asterisks (*P < 0.05 and **P < 0.01 in t test).

  • Fig. 8 Abnormal features of femoral growth plates in chondrocyte-specific Trpm7-deficient mice.

    (A) Ca2+ fluctuations in round chondrocytes in femoral bone slices prepared from chondrocyte-specific Trpm7 knockout [Trpm7fl/fl (11Enh-Cre+/−)] and control [Trpm7fl/fl (11Enh-Cre−/−)] embryos at E17.5. Recordings were performed in the absence and presence of the TRPM7 activator naltriben. Typical recording traces from individual Ca2+ fluctuation–positive (red, purple, and green) and Ca2+ fluctuation–negative (black) cells are shown. The proportion of cells that showed Ca2+ fluctuations and the amplitudes of the fluctuations were quantified and statistically analyzed. (B) Mid-longitudinal sections of femoral specimens from Trpm7fl/fl (11Enh-Cre+/−) and Trpm7fl/fl (11Enh-Cre−/−) embryos show the zones of round (R), columnar (C), and hypertrophic (H) chondrocytes. Scale bar, 1 mm. Quantification of total bone length and sizes of the entire growth plate (Total) and each chondrocyte zone. (C) Representative images of the round, columnar, and hypertrophic cell zones in femurs from Trpm7fl/fl (11Enh-Cre+/−) and Trpm7fl/fl (11Enh-Cre−/−) embryos. Scale bar, 100 μm. Cell sizes in the growth plate zones were measured and statistically analyzed. (D) Immunoblotting and quantification of CaMKII autophosphorylation in femoral growth plates of Trpm7fl/fl (11Enh-Cre+/−) and Trpm7fl/fl (11Enh-Cre−/−) embryos. (E) RT-PCR analysis of the indicated transcripts in growth plates of Trpm7fl/fl (11Enh-Cre+/−) and Trpm7fl/fl (11Enh-Cre−/−) embryos. Data represent means ± SEM, and the numbers of mice and cells examined are shown in parentheses. Statistical differences between the genotypes are indicated with asterisks (*P < 0.05 and **P < 0.01 in t test). (F) Model of signaling downstream of TRPM7-mediated Ca2+ entry in chondrocytes. This scheme is proposed on the basis of the similarity of defects between Trpm7-deficient and CaMKII inhibitor–treated bones and is further supported by insufficient CaMKII autophosphorylation and altered gene expression in Trpm7-deficient growth plates.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/576/eaaw4847/DC1

    Fig. S1. Ca2+ imaging in hypertrophic and perichondrial cells and pharmacological characterization of Ca2+ fluctuations.

    Fig. S2. Gene expression analysis and effects of TRPM7 modulators on growth plate chondrocytes.

    Fig. S3. Conditional knockout of Trpm7.

    Fig. S4. Effects of PLC and BK channel modulators on naltriben-facilitated Ca2+ fluctuations.

    Fig. S5. Characterization of facilitated Ca2+ fluctuations under store-depleted conditions.

    Fig. S6. Gene expression analysis in cultured Trpm7-deficient metatarsal bones.

    Fig. S7. Effects of CaMKII and CaN inhibitors on cultured metatarsal bones.

    Fig. S8. Generation and characterization of chondrocyte-specific Trpm7-deficient mice.

    Fig. S9. Extracellular Mg2+ and cultured Trpm7-deficient bones.

    Table S1. PCR primers used in the study.

  • This PDF file includes:

    • Fig. S1. Ca2+ imaging in hypertrophic and perichondrial cells and pharmacological characterization of Ca2+ fluctuations.
    • Fig. S2. Gene expression analysis and effects of TRPM7 modulators on growth plate chondrocytes.
    • Fig. S3. Conditional knockout of Trpm7.
    • Fig. S4. Effects of PLC and BK channel modulators on naltriben-facilitated Ca2+ fluctuations.
    • Fig. S5. Characterization of facilitated Ca2+ fluctuations under store-depleted conditions.
    • Fig. S6. Gene expression analysis in cultured Trpm7-deficient metatarsal bones.
    • Fig. S7. Effects of CaMKII and CaN inhibitors on cultured metatarsal bones.
    • Fig. S8. Generation and characterization of chondrocyte-specific Trpm7-deficient mice.
    • Fig. S9. Extracellular Mg2+ and cultured Trpm7-deficient bones.
    • Table S1. PCR primers used in the study.

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