Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: Phosphorylation of MSK1 via the p38 kinase pathway upon BMP-6 stimulation in ATDC5 cells. A , phosphorylation of MSK1 (Ser376) and MSK1 (Ser360) by BMP-6. ATDC5 cells were stimulated with 25 ng/ml BMP-6 for the indicated times. The total cell lysates were then used for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-Smad1/5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6. B , Inhibition of BMP-6-induced MSK1 phosphorylation by dorsomorphin, a BMP type I receptor kinase inhibitor. ATDC5 cells were pretreated with 10 μM dorsomorphin for 1 h. Thereafter, cells were stimulated with 25 ng/ml BMP-6 for the indicated times. The total cell lysates were used for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of dorsomorphin. C , Involvement of the ERK pathway in the phosphorylation of MSK1 by BMP-6. ATDC5 cells were pretreated with 20 μM U0126 1 h before stimulation with 25 ng/ml BMP-6 for the indicated times. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of U0126. D , decrease in BMP-6-induced phosphorylation of MSK1 in the presence of the p38 kinase inhibitor. ATDC5 cells pretreated with 10 μM SB203580, a p38 kinase inhibitor, for 1 h were stimulated with 25 ng/ml BMP-6 for the indicated times. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), p38, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth , and bottom panels , respectively. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of SB203580. A-D , white and black arrowheads indicate phospho-MSK1 (Ser376)-specific and nonspecific bands, respectively. E , decrease in BMP-6-mediated phospho-CREB (Ser133) expression by the MSK1 kinase inhibitor, SB747651A. ATDC5 cells were cultured with 10 μM SB747651A for 1 h. Prior to preparation of the total lysates, 25 ng/ml BMP-6 was added to the culture media for the indicated times. The total expression levels of phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth , and bottom panels , respectively. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells with BMP-6 in the absence of SB747651A. F , chondrogenic differentiation of ATDC5 cells by BMP-6 in the presence of kinase inhibitors. ATDC5 cells cultured at a high density were stimulated with 25 ng/ml BMP-6 in the presence (+) or absence (−) of kinase inhibitors (20 μM U0126, 10 μM SB203580, 10 μM SB747651A, or 10 μM dorsomorphin). Two weeks later, the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the 1-h treatment of cells with BMP-6 alone. G , representative images of ex vivo chondrogenic differentiation of mouse metatarsal bones induced by BMP-6 in the presence of kinase inhibitors. Each mouse metatarsal bone was cultured with or without 25 ng/ml BMP-6 for 7 days, either in the absence or presence of kinase inhibitors (20 μM U0126, 10 μM SB203580, 10 μM SB747651A, or 10 μM dorsomorphin). Then, each bone was stained with Alcian blue and Alizarin Red S. Two representative images are shown in each treatment. a , proliferative chondrocytes; b , clear zone represented as hypertrophic chondrocytes; c , chondrocyte matrix calcified by mature hypertrophic chondrocytes stained with alizarin red S. H , relative area of chondrocyte differentiation in mouse metatarsal bones induced by BMP-6 in the presence of kinase inhibitors. The total areas of chondrocyte differentiation (a + b + c) were measured. The total areas of chondrocyte differentiation (a + b + c) were measured using ImageJ. Asterisks indicate significant differences. BMP, bone morphogenetic protein; CREB, cAMP-response element-binding protein; MSK, mitogen- and stress-activated protein kinase; ND; not determined.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Phospho-proteomics, Western Blot, Expressing, Inhibition, Cell Culture, Staining, Ex Vivo, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: I nflu ence of MSK1 deficiency on BMP-6–mediated chondrogenic differentiation. A , expression of MSK1 in ATDC5 cells. Total lysates from each transformant were prepared for Western blot analyses. The total expression levels of MSK1 and β-actin are indicated in the upper and lower panels , respectively. White and black arrowheads indicate MSK1-specific and nonspecific bands, respectively. The intensity of the band for MSK1 was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to cells carrying shControl. B , chondrogenic differentiation of ATDC5 cells with decreased expression of MSK1 in the presence of BMP-6. The cells were seeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying shControl with BMP-6 for 14 days. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Expressing, Western Blot, Staining, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: R equi rement of N-terminal and C-terminal kinase activities of MSK1 for the induction of chondrocyte differentiation. A , depiction of MSK1 and its mutants used in . B , phosphorylation of MSK1 or its mutants in ATDC5 cells stimulated with BMP-6. To induce the expression of ectopic MSK1 or its mutants in ATDC5 cells, cells were cultured in the presence of tetracycline for 16 h. Thereafter, the ATDC5 transformants were stimulated with 25 ng/ml BMP-6 for 1 h. The total expression levels of phospho-MSK1 (Ser376), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensities of the bands for phospho-MSK1 (Ser376) and phospho-CREB (Ser133) were normalized to the intensities of the bands corresponding to MSK1 and β-actin, respectively. The relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6 alone. C , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or its mutants in the presence of BMP-6. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were reseeded at a high density and stimulated with 25 ng/ml BMP-6. Seven ( upper photos ) or 14 days later ( lower photos ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 alone for 14 days. D and E , According to C , ATDC5 cells expressing WT MSK1 or MSK1 (D195A/D565A) were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, the total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( D ) and collagen10a1 mRNAs ( E ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; CTKD, C-terminal kinase domain; HM, hydrophobic motif; MSK, mitogen- and stress-activated protein kinase; ND, not determined; NTKD, N-terminal kinase domain; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Staining, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: M SK1 m utants lacking phosphorylation site targeted by p38 kinase and C-terminal kinase of MSK1. A , depiction of MSK1 and its mutants used in and . B , phosphorylation of MSK1 or its mutants in ATDC5 cells stimulated with BMP-6. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were stimulated with 25 ng/ml BMP-6 for 1 h and their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. C , comparison between serine-360 and threonine-581 in MSK1 as targeted amino acid residues for p38 kinase. After expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in B , the cells were stimulated with 25 ng/ml BMP-6 for 1 h and then their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), phospho-MSK1 (Ser360), MSK1, phospho-CREB (Ser133), phospho-p38 (Thr180/Tyr182), p38, phospho-Smad1/5, Smad5, and β-actin are indicated in the upper, second, third, fourth, fifth, sixth, seventh, eighth, and bottom panels , respectively. A black arrow and a white arrowhead indicate ectopic and endogenous phospho-MSK1 (Ser376)-specific band, respectively. A black arrowhead indicates nonspecific bands. The intensity of the bands for phospho-MSK1 (Ser376) and phospho-MSK1 (Ser360) was normalized to the intensity of the band corresponding to MSK1. The intensity of the band for phospho-CREB (Ser133) was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. D , phosphorylation of threonine at position 581 in MSK1 upon BMP-6 stimulation. As described in B , cell lysates were prepared from each transformant. The total expression levels of phospho-MSK1 (Thr581), MSK1, phospho-Smad1/5, and β-actin are indicated in the upper, second, third, and bottom panels , respectively. The intensity of the band for phospho-MSK1 (T581) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells carrying WT MSK1 with BMP-6. E , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or its mutants in the presence of BMP-6. After the expression of WT MSK1 or its mutants in ATDC5 cells using tetracycline as described in C , the cells were reseeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 alone for 14 days. F and G , expression of aggrecan and collagen 10a1 mRNAs in ATDC5 cells and their transformants. According to , D and E , ATDC5 cells expressing WT MSK1 or its mutants were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( F ) and collagen10a1 mRNAs ( G ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; CREB, cAMP-response element-binding protein; MSK, mitogen- and stress-activated protein kinase; ND, not determined; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Phospho-proteomics, Expressing, Western Blot, Comparison, Staining, Cell Culture, Binding Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: Req u irement of autophosphorylation of Ser 376 in MSK1 for its kinase activity upon BMP-6 stimulation. A , chondrogenic differentiation of ATDC5 cells carrying WT MSK1 or MSK1 (S376A) mutant in the presence of BMP-6. After the expression of WT MSK1 or MSK1 (S376A) mutant in ATDC5 cells using tetracycline as described in C , the cells were reseeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying WT MSK1 with BMP-6 for 14 days. B and C , expression of aggrecan and collagen10a1 mRNAs in ATDC5 cells and their transformant. According to , D and E , ATDC5 cells expressing the WT MSK1 or its mutant were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( B ) and collagen10a1 mRNAs ( C ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Activity Assay, Mutagenesis, Expressing, Staining, Cell Culture, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

doi: 10.1016/j.jbc.2024.107806

Figure Lengend Snippet: Effect of the BMP/Smad-dependent pathway on BMP-6–mediated chondrogenic differentiation. A , expression of Smad4 in ATDC5 cells. Total lysates from each transformant were prepared for Western blot analyses. The total expression levels of Smad4 and β-actin are indicated in the upper and lower panels , respectively. The intensity of the band for Smad4 was normalized to the intensity of the band corresponding to β-actin. Relative intensity was calculated with respect to cells carrying shControl. B , chondrogenic differentiation of ATDC5 cells with decreased expression of Smad4 in the presence of BMP-6. The cells were seeded at a high density and then stimulated with 25 ng/ml BMP-6. Seven ( upper panels ) or 14 days later ( lower panels ), the cells were stained with Alcian blue. Representative images are shown. The intensity of the area stained with Alcian blue was measured using ImageQuant TL. The relative intensity of the stained area was calculated with respect to the cells carrying shControl with BMP-6 for 14 days. C and D , expression of aggrecan and collagen10a1 mRNA in ATDC5 cells with Smad4 knockdown. According to Fig. 2 , D and E , ATDC5 cells with decreased expression of Smad4 were cultured with 25 ng/ml BMP-6 for 14 days. Thereafter, total mRNAs from each transformant were prepared for qPCR to detect aggrecan ( C ) and collagen10a1 mRNAs ( D ). All values represent means ± SDs (n = 3). Asterisks indicate significant differences. E , effect of Smad4 deficiency on BMP-6–induced p38 phosphorylation. The cells were stimulated with 25 ng/ml BMP-6 for 1 h and then their total lysates were prepared for Western blot analyses. The total expression levels of phospho-MSK1 (Ser376), MSK1, phospho-p38 (Thr180/Tyr182), phospho-Smad1/5, and β-actin are indicated in the upper, second, third, fourth, and bottom panels , respectively. The intensity of the band for phospho-MSK1 (S376) was normalized to the intensity of the band corresponding to MSK1. Relative intensity was calculated with respect to the 1-h treatment of cells carrying shControl with BMP-6. BMP, bone morphogenetic protein; MSK, mitogen- and stress-activated protein kinase; ND, not determined; qPCR, quantitative PCR.

Article Snippet: The intensity of each band was measured using ImageQuant TL analysis software (Cytiva) after each gel image was captured with the ImageQuant LAS 4000 mini (Amersham).

Techniques: Expressing, Western Blot, Staining, Knockdown, Cell Culture, Phospho-proteomics, Real-time Polymerase Chain Reaction