Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Sep;28(18):5785-94.
doi: 10.1128/MCB.00245-08. Epub 2008 Jul 14.

Retinoic acid utilizes CREB and USF1 in a transcriptional feed-forward loop in order to stimulate MKP1 expression in human immunodeficiency virus-infected podocytes

Ting-Chi Lu  1 , Zhaohui Wang, Xiaobei Feng, Peter Chuang, Wei Fang, Yibang Chen, Susana Neves, Avi Maayan, Huabao Xiong, Yusen Liu, Ravi Iyengar, Paul E Klotman, John Cijiang He
Affiliations

Retinoic acid utilizes CREB and USF1 in a transcriptional feed-forward loop in order to stimulate MKP1 expression in human immunodeficiency virus-infected podocytes

Ting-Chi Lu et al. Mol Cell Biol. 2008 Sep.

Abstract

Nef-induced podocyte proliferation and dedifferentiation via mitogen-activated protein kinase 1,2 (MAPK1,2) activation plays a role in human immunodeficiency virus (HIV) nephropathy pathogenesis. All-trans retinoic acid (atRA) reverses the HIV-induced podocyte phenotype by activating cyclic AMP (cAMP)/protein kinase A (PKA) and inhibiting MAPK1,2. Here we show that atRA, through cAMP and PKA, triggers a feed-forward loop involving CREB and USF1 to induce biphasic stimulation of MKP1. atRA stimulated CREB and USF1 binding to the MKP1 gene promoter, as shown by gel shifting and chromatin immunoprecipitation assays. CREB directly mediated the early phase of atRA-induced MKP1 stimulation; whereas the later phase was mediated by CREB indirectly through induction of USF1. These findings were confirmed by a reporter gene assay using the MKP1 promoter with mutation of CRE or Ebox binding sites. Consistent with these findings, the biological effects of atRA on podocytes were inhibited by silencing either MKP1, CREB, or USF1 with small interfering RNA. atRA also induced CREB phosphorylation and MKP1 expression and reduced MAPK1,2 phosphorylation in kidneys of HIV type 1-infected transgenic mice. We conclude that atRA induces sustained activation of MKP1 to suppress Nef-induced activation of the Src-MAPK1,2 pathway, thus returning the podocyte to a more differentiated state. The mechanism involves a feed-forward loop where activation of one transcription factor (TF) (CREB) leads to induction of a second TF (USF1).

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
atRA-induced MKP1 gene expression is biphasic and requires cAMP/PKA activation. (A) Podocytes were stimulated with 1 μM atRA for the indicated time intervals. Total RNA was isolated for real-time PCR of MKP1. The ratios of MKP1/tubulin mRNA levels were compared with those for control (untreated) cells. *, P < 0.01 compared to unstimulated cells (n = 3). (B) HIV-infected podocytes were pretreated with Rp-cAMP (100 μM) for 2 h and then stimulated with atRA (1 μM) for the time intervals indicated. Total RNA was isolated for real-time PCR of MKP1 as described above (n = 3). (C) Podocytes were stimulated with 1 μM atRA for the indicated time intervals. Cell lysates were prepared for immunoblot analysis using anti-phospho-MAPK1,2 (pMAPK1,2), total MAPK1,2 (T-MAPK1,2), MKP1, and β-actin antibodies. (D) HIV-infected podocytes were pretreated with Rp-cAMP at 100 μM for 2 h and then stimulated with atRA (1 μM) at the indicated time points. Immunoblotting was performed for phosphor-MAPK1,2, total MAPK1,2, MKP1, and β-actin. (E) HIV-infected podocytes were transfected with siRNA for MKP1 and then stimulated with atRA (1 μM) at the indicated time points. Immunoblotting was performed for phosphor- and total MAPK1,2, MKP1, and β-actin. All experiments were repeated at least three times, and representative blots are shown.
FIG. 2.
FIG. 2.
CREB and USF1 mediate the effects of atRA on MKP1 stimulation. (A) Control and HIV-infected podocytes were pretreated with either Rp-cAMP (100 μM) or control vehicle for 60 min and then incubated with atRA (1 μM) for the times indicated. Cell lysates were obtained for immunoblot analysis using phosphor- (P-CREB) and total CREB (T-CREB) antibodies. (B1) EMSA was performed for CREB as described in Materials and Methods. Anti-CREB antibody (Ab) (Cell Signaling) was used for the supershift experiment. DMSO, dimethyl sulfoxide. (B2) Cells were stimulated with atRA (1 μM) for the indicated times. Cell lysates were used for immunoprecipitation (IP) with monoclonal anti (α)-CBP antibody (Santa Cruz). The immunoprecipitates and total cell lysates were used for immunoblots (IB) using anti-CREB antibody (Santa Cruz). (C) EMSA was performed for USF1 as described in Materials and Methods. Anti-USF1 antibody (Santa Cruz) was used for the supershift experiment. (D) Podocytes were transfected with either K-CREB or control vector as described in Materials and Methods. Cell lysates were prepared for immunoblot analysis of total CREB in order to verify overexpression of K-CREB. Representative blots of three independent experiments are shown. (E) Podocytes were transfected with siRNA for USF1 or control (CL) siRNA. Immunoblotting (E1) and real-time PCR (E2) were performed to confirm suppression of USF1 expression, *, P < 0.001 (n = 3). (F) Podocytes were transfected with K-CREB, siRNA for USF1, or control siRNA for 3 days. Cells were stimulated with atRA (1 μM) for different time intervals as indicated. Total RNA was isolated for real-time PCR for MKP1 as described in Materials and Methods. The ratios of MKP1/tubulin mRNA levels in atRA-stimulated cells were compared with those for control cells. The means of three independent experiments are shown. The stimulation by atRA relative to that for the control is shown, *, P < 0.01 for K-CREB-transfected cells compared to mock-transfected cells; **, P < 0.01 for siRNA for USF1 siRNA-transfected cells compared to mock-transfected cells. (G) Podocytes were transfected with K-CREB, siRNA for USF1, or control siRNA for 3 days. Cells were stimulated with atRA (1 μM) for different time intervals as indicated. Cell lysates were prepared for immunoblot analysis with MKP1 antibody. All experiments were repeated at least three times, and the representative blots are shown.
FIG. 3.
FIG. 3.
The role of CREB and USF1 in mediating the stimulatory effects of atRA on MKP1 was further confirmed. (A) Podocytes were transfected with either control (CL) siRNA or siRNA specific for CREB for 3 days. CREB expression was determined in these cells by immunoblotting (A1) and real-time PCR (A2) (n = 3; *, P < 0.01). (B) Podocytes were transfected with either K-CREB or siRNA for CREB or control vector for 3 days and then stimulated with atRA for the indicated times. Real-time PCR was performed in these cells to determine the ratio of MKP1/tubulin mRNA levels (n = 3; *, P < 0.01). (C) ChIP analysis. Podocytes were stimulated with atRA or control vehicle for 30 min for CREB and 90 min for USF1. The MKP1 promoter-specific sequence was immunoprecipitated by anti-CREB and anti-USF1 antibodies but not by control IgG. The representative blots of three independent experiments are shown. (D) Podocytes were transfected with WT or mutant MKP1 reporter genes for 3 days. Cells were then stimulated with atRA (1 μM) for the indicated times. Cells were harvested for total RNA isolation and real-time PCR for luciferase mRNA levels (n = 3; *, P < 0.01). (E) Podocytes were transfected with WT or mutant MKP1 reporter genes for 3 days. Cells were stimulated by atRA for 6 h, and then luciferase activity was determined as described in Materials and Methods (n = 3; *, P < 0.05). C, control.
FIG. 4.
FIG. 4.
atRA activates CREB, which in turn activates USF1 through stimulation of its expression. (A) HIV-infected podocytes were pretreated with CHX (5 mM) for 30 min and then stimulated with atRA (1 μM) or dimethyl sulfoxide (DMSO) for the indicated time intervals. Total RNA was isolated for real-time PCR for MKP1 (P < 0.01; n = 3). (B) HIV-infected podocytes were treated with CHX or atRA as for panel A. Nuclear proteins were isolated for EMSA. (C) Podocytes were treated with atRA (1 μM) for the indicated time intervals. Cell lysates with or without treatment with calf intestine alkaline phosphatase (Invitrogen) were used for immunoblotting for USF1 after running on a gradient gel. Total CREB was used as loading control for nuclear proteins. (D) Podocytes were pretreated with CHX (5 mM) for 30 min and then treated with atRA as for panel A. Immunoblotting was performed for USF1 and CREB as for panel C. (E) Podocytes were transfected with K-CREB for 3 days and then treated with atRA as for panel A. Immunoblotting was performed for USF1 and CREB as for panel C. (F) Podocytes were treated with Rp-cAMP (100 μM) for 1 h and then treated with atRA as for panel A. Immunoblotting were performed for USF1 and CREB as for panel C. The representative blots of at least three independent experiments are shown here.
FIG. 5.
FIG. 5.
MKP1 mediates the effects of atRA on podocytes. HIV-infected podocytes were transfected with either control vector or MKP1 (A to C) or transfected with a control oligonucleotide or siRNA against MKP1 (D to F) for 3 days. Cells were then stimulated with atRA at 1 μM for 1 to 5 days. Real-time PCR was used to assess MKP1 expression levels (A and D). Cell number was assessed at days 1, 3, and 5 (B and D), and real-time PCR was performed using primers for synaptopodin (C and F). All experiments were repeated at least three times, *, P < 0.01.
FIG. 6.
FIG. 6.
Role of CREB and USF1 in mediating the effects of atRA on podocytes in vitro. HIV-1-infected podocytes were transfected with control vector, K-CREB, siRNA for CREB1, siRNA for USF1, or control siRNA for 3 days and then were incubated with or without atRA (1 μM) for 1 to 3 days. (A) Cell number was assessed in these conditions at day 3. The means ± standard deviations of three independent experiments performed in triplicate are shown, *, P < 0.001; **, P < 0.05. DMSO, dimethyl sulfoxide. (B) Real-time PCR of synaptopodin was performed in these cells with or without atRA stimulation for 1 day. *, P < 0.01.
FIG. 7.
FIG. 7.
atRA induces CREB phosphorylation and MKP1 expression in vivo. HIV-1-infected transgenic mice were injected with atRA or vehicle alone (three mice in each group) as described in Materials and Methods. After perfusion with phosphate-buffered saline containing phosphatase inhibitors, the cortex of the kidney was removed and isolated glomeruli were used for immunoblotting using specific antibodies for phosphor (p)- and total (T) CREB, MKP1, USF1, phosphor- and total MAPK1,2, and β-actin.
FIG. 8.
FIG. 8.
Schematic of cross talk between cAMP/PKA and MAPK1,2 downstream of atRA signaling in HIV-infected podocytes. Nef, an HIV accessory protein, interacts with Src, leading to Ras/C-Raf/MAPK1,2 phosphorylation in podocytes. This sustained MAPK1,2 phosphorylation contributes to podocyte proliferation and dedifferentiation. atRA stimulates intracellular cAMP production, which activates PKA, leading to CREB phosphorylation. CREB directly mediated the early phase of atRA-induced MKP1 stimulation, whereas the later phase was mediated indirectly by CREB through induction of USF1 expression. Through this feed-forward gene-regulatory motif, atRA induced sustained upregulation of MKP1 to suppress HIV-induced activation of the MAPK1,2 pathway, leading to podocyte differentiation and growth arrest.
See this image and copyright information in PMC

References

    1. Abraham, S. M., T. Lawrence, A. Kleiman, P. Warden, M. Medghalchi, J. Tuckermann, J. Saklatvala, and A. R. Clark. 2006. Antiinflammatory effects of dexamethasone are partly dependent on induction of dual specificity phosphatase 1. J. Exp. Med. 2031883-1889. - PMC - PubMed
    1. Allenby, G., M. T. Bocquel, M. Saunders, S. Kazmer, J. Speck, M. Rosenberger, A. Lovey, P. Kastner, J. F. Grippo, P. Chambon, et al. 1993. Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proc. Natl. Acad. Sci. USA 9030-34. - PMC - PubMed
    1. Awazu, M., K. Ishikura, et al. 1999. Mechanisms of mitogen-activated protein kinase activation in experimental diabetes. J. Am. Soc. Nephrol. 10738-745. - PubMed
    1. Awazu, M., S. Omori, et al. 2002. MAP kinase in renal development. Nephrol. Dial. Transplant. 17(Suppl. 9)5-7. - PubMed
    1. Barisoni, L., L. A. Bruggeman, P. Mundel, V. D. D'Agati, and P. E. Klotman. 2000. HIV-1 induces renal epithelial dedifferentiation in a transgenic model of HIV-associated nephropathy. Kidney Int. 58173-181. - PubMed

Publication types

MeSH terms

LinkOut - more resources