HIVAN is the most common kidney disease in HIV-seropositive patients, and specific treatment for this disease is unavailable. While antiretroviral therapy ameliorates the disease, adjunctive treatment targeted to specific pathogenic pathways induced by HIV-1 awaits further development. Recently, we found that atRA induces HIV-infected podocytes to differentiate and inhibits HIV-induced podocyte proliferation in vitro (14
). atRA also reduces proteinuria and glomerulosclerosis in vivo in HIV-1 transgenic mice (14
). Inhibition of MAPK1,2 phosphorylation is a critical part of the mechanism (14
). In the current study, we have explored the molecular and cellular mechanisms responsible for MAPK1,2 inhibition as well as the impact of this pathway on disease phenotype. We conclude that atRA inhibits MAPK1,2 phosphorylation through stimulation of MKP1 based on the following findings: (i) the time course of MKP1 stimulation by atRA at protein levels correlates with the time course of MAPK1,2 phosphorylation levels, (ii) silencing MKP1 expression by siRNA causes sustained MAPK1,2 phosphorylation, and (iii) atRA did not induce MKP2 expression.
atRA has been shown to cause increased MKP1 expression in other cell types (30
). The mechanism, however, has remained unclear. Expression of MKP1 can be induced by activation of PKA, protein kinase C, or MAPKs (MAPK1,2, p38, and JNK) in response to multiple stimuli (8
). While upstream events are fairly well known, the identity of downstream, target transcription factors has been largely unknown. We show that atRA induces MKP1 gene expression in podocytes through PKA-mediated CREB and USF1 activation. The early phase of MKP1 stimulation is regulated by CREB directly; the late phase of stimulation is controlled by CREB indirectly through induction of USF1 expression and activation. These findings were confirmed by our reporter gene assay using a mutant MKP1 promoter with deletion of either CRE or Ebox. Furthermore, both CREB and USF1 can bind to cis
elements of the MKP1 gene promoter, as shown by our ChIP experiments as well as by others (32
Our findings suggest that a feed-forward motif is required for sustained MKP induction. Since CREB acts upstream of this feed-forward loop, inhibition of CREB by K-CREB or siRNA completely inhibited MKP1 stimulation and cell proliferation. USF1 mediated only the late phase of MKP1 stimulation and produced less-sustained stimulation of MKP1 expression. Therefore, inhibition of USF1 only partially reduced the antiproliferative effects of atRA. Our data suggest that this feed-forward loop (CREB-USF1) is required for sustained MKP1 expression and for the full effects of atRA on podocytes. Of note, while atRA induced a clear biphasic stimulation of MKP1 at the mRNA level, immunoblotting demonstrated monophasic and sustained expression of MKP1 at the protein level. Inhibition of USF1 reduced the time frame of MKP1 activation by atRA.
CREB is phosphorylated by several protein kinases, including PKA and MAPK1,2 (27
). We have already demonstrated the beneficial effects of atRA in HIV-1-infected transgenic mice (14
). We found here that atRA induced CREB phosphorylation through activation of PKA. It was recently reported that atRA induces neurite outgrowth in PC12 cells through CREB phosphorylation and that this is independent of the retinoic acid response elements (9
). While the importance of CREB in neuronal differentiation is well documented, the role of CREB in podocytes and in kidney disease has never been studied. We found here that inhibition of CREB activity diminished the effects of atRA on podocytes, indicating that CREB acts downstream of atRA in maintaining the podocyte in a differentiated state. Our data indicate a new pathway in which CREB stimulates the expression of the MKP1 gene, leading to podocyte differentiation. atRA also induced CREB phosphorylation in vivo, which is associated with increased MKP1 expression and reduced MAKP1,2 phosphorylation. This is the first study to show that atRA induces a CREB signaling pathway in vivo.
USF1 is a ubiquitously expressed transcription factor controlling several critical genes in lipid and glucose metabolism (20
). Of some 40 genes regulated by USF1, several are involved in the molecular pathogenesis of cardiovascular disease. Polymorphisms of this gene are associated with kidney disease as well (10
). The USF1 gene promoter in the podocin (NPHS2) gene promoter has been shown to affect gene expression. Podocin (NPHS2) expression in podocytes is associated with variable degrees of proteinuria and progression to renal failure in different glomerular diseases (7
). We found here that USF1 mediates the protective effects of atRA on podocytes by stimulation of the MKP1 gene along with CREB. Dual regulation of genes with USF1 and other transcription factors is well known. It has been reported that USF1 and AP-2b cooperatively regulate the human lipocalin-type prostaglandin D synthase gene in TE671 cells (12
). USF1/2 and CREB together mediate Helicobacter pylori
-dependent COX-2 gene transcription via a MEK/extracellular signal-regulated kinase-dependent pathway (18
). Furthermore, both CREB and USF1 contribute to Ca2+
signal-mediated activation of brain-derived neurotrophic factor gene promoter 1 expression in rat cortical neurons in culture (37
). However the role of network topology in these other cases is not known. Furthermore, the temporal regulation of genes by different transcription factors is not well studied. Only one study has reported that UTP stimulates osteopontin expression via coordinated regulation of the NF-κB, USF, and AP-1 signaling pathways (31
). While NF-κB mediates early activation (15 min), USF1/2 and AP-1 mediate late-phase activation (1 h). The authors speculate that NF-κB mediates the early phase because phosphorylation is sufficient for its activation whereas activation of USF1 and AP1 requires de novo synthesis of cofactors. In contrast, our study provides a mechanistic understanding of the relationship between CREB and USF1. atRA-induced CREB activation mediated increases in USF1 protein levels that were required for enhancement of USF1 activity in podocytes. Therefore, the effect of USF1 is delayed compared to that of CREB, which is activated by direct phosphorylation. Thus our data provide evidence that a feed-forward loop is required for sustained increases in MKP levels.
Our data also support a role for this increase in MKP1 in mediating the protective effects of atRA on podocytes. MKP1 has been shown to be important in the innate immune responses and plays a critical role in suppressing endotoxin-induced shock (43
). A recent study suggests that MKP1 contributes to the anti-inflammatory effects of dexamethasone both in vitro and in vivo (1
). MKP1 has been reported to mediate the inhibitory effects of atRA on the hypertrophic growth of cardiomyocytes (30
). The role of MKP1 in kidney disease has also been determined. It has been shown that connective tissue growth factor promoters activate mesangial cell survival via upregulation of MKP1 (39a
). In kidneys of diabetic rats, MKP1 expression increases in parallel with p38 at the early phase (19
). However, other studies suggest that MKP1 is suppressed in kidneys with diabetic nephropathy, leading to increased MAPK phosphorylation (2a
). These controversial findings require further studies to clarify. MKP1 expression may change at different stages of kidney disease. It is very interesting that MAPK (extracellular signal-regulated kinase, p38, and JNK) and MKP1 are highly expressed in proliferative cells in renal development (2b
). An increase of MKP1, possibly induced by MAPK, may not be strong enough to suppress MAPK activation. The temporal relationship between MAPK activation and MKP1 activation in vivo remains to be determined. Our study indicates that MKP1 plays a key role in mediating the effects of atRA to inhibit proliferation and induce differentiation of podocytes infected by HIV. We also show that these effects are mediated through the inhibition of MAPK activation.
In conclusion, the current study shows that atRA, by triggering a feed-forward gene regulatory motif between CREB and UFS1, induced sustained upregulation of MKP1 to suppress HIV Nef-induced activation of the Src-MAPK1,2 pathway and returned the podocyte to a more differentiation state (Fig. ).
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 (more ...)