Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. A potential marker transcript for chronic pathologic conditions, such as diabetic nephropathy. Possible role in the response to persistent stress

Abstract

Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extracellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH(2)-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences.

Fig. 1. Transcriptional induction of gene 33 in response to osmotic stress

Cultured (passage 3) rat renal mesangial cells were treated with 500 mM sorbitol for 15 min, at which time the medium was returned to iso-osmotic conditions. Samples were removed after the sorbitol treatment or for various times after restoration of normal conditions and assayed for gene 33 expression by Northern blot.

Fig. 2. Transcriptional induction of gene 33 in response to unilateral nephrectomy and streptozotocin-induced diabetes: gene 33 expression as a marker for diabetic nephropathy

Male rats were made diabetic upon injection of streptozotocin as described under “Experimental Procedures.” Control animals were injected with water. A, transcription pattern in early diabetes. At the times indicated after streptozotocin injection, one kidney was removed from control or diabetic animals, and RNA was prepared and analyzed for gene 33, c-jun, and c-fos expression by Northern blot. The remaining contralateral kidney was removed later, at the indicated time, to assess the effects of unilateral nephrectomy on gene 33 expression in an identical manner. The numbers indicate the hours elapsed between the treatment (nephrectomy, streptozotocin, or both) and harvest of the kidney. B, same as A, except that the animals were allowed to proceed to frank diabetic nephropathy (5 weeks post-streptozotocin injection). In these assays, unilateral nephrectomy was not performed, and only the effects of diabetes on gene expression were tested. The blots were probed for glyceraldehyde-3-phosphate dehydrogenase (gapdh) expression as a loading control.

Fig. 3. Transcriptional induction of gene 33 in response to vasoactive peptides, calcium ionophore, and mechanical strain: Induction by mechanical strain requires SAPK activity

A, rat renal mesangial cells were treated with endothelin (ET-1), angiotensin-II (A-II), or calcium ionophore (A23187) as indicated (see “Experimental Procedures”). gene 33 induction was determined by Northern blot. gapdh expression served as a loading control. B, pulmonary A549 cells were infected with the indicated recombinant adenoviruses at the indicated Pfu/cell and, after 48 h, subjected to mechanical strain (see “Experimental Procedures”). gene 33 induction was determined by Northern blot. gapdh expression served as a loading control.

Fig. 4. Structure of the Gene 33 polypeptide

The top diagram indicates the modular structure of Gene 33 which is suggestive of an adapter protein, since no apparent catalytic sequences are present. The bottom panel compares the putative domains of Gene 33 with the cognate domains in established signaling proteins. Conserved amino acid residues are indicated with colons; vertical lines indicate identical residues. Inferences concerning possible functional characteristics are based on sequence homology.

Fig. 5. Gene 33 is a cytosolic protein that interacts with 14-3-3ζ and, in a GTP-dependent manner, with Cdc42Hs

A, cytosolic localization of Gene 33. NIH3T3 cells were transfected with FLAG-Gene 33. Cells were stained with anti-FLAG and fluorescein isothiocyanate-labeled anti-mouse. Gene 33 was detected by immunofluorescence. The arrowhead indicates the nucleus. B, interaction with 14-3-3ζ. 293 cells were transfected with GST-14-3-3ζ and FLAG-Gene as indicated. GST pull-downs and anti-FLAG immunoprecipitates (IPs) were prepared and subjected to SDS-PAGE and reciprocal immunoblotting with anti-GST and anti-FLAG to detect coprecipitated proteins. C, in vivo binding of Gene 33 to Cdc42Hs. 293 cells were transfected with GST-Gene 33 and FLAG-V12-Cdc42Hs. Anti-FLAG-Cdc42Hs immunoprecipitates (IP) were prepared and subjected to washing with progressively higher concentrations of LiCl as indicated in the top panel, followed by SDS-PAGE and immunoblotting (IB) with anti-GST to detect bound Gene 33. Expression blots are shown in the bottom panel. D, the Gene 33/Cdc42Hs interaction requires the Gene 33 CRIB motif. Cells were transfected with FLAG-Cdc42Hs (V12) plus either GST-Gene 33 or HA-Gene 33 (aa 61–459). Cdc42Hs was immunoprecipitated with anti-FLAG and subjected to SDS-PAGE and immunoblotting with anti-GST or anti-HA as indicated. Crude lysates were blotted with the indicated antibodies to judge expression of the transfected proteins. E, in vitro GTP-dependent binding of Gene 33 to Cdc42Hs. Bacterially expressed GST-Cdc42Hs was loaded with either GTP-γS or GDP-βS as indicated (GTP or GDP, respectively) and incubated with extracts of 293 cells that had been transfected with Gene 33 or PAK1 (FLAG-tagged) expressed at equal levels (left) or with Gene 33 in excess (right). Crude 293 cell extracts, indicated in the figure, were subjected to SDS-PAGE and blotted with anti-FLAG to detect expression of Gene 33 or PAK1. Cdc42Hs beads were washed and subjected to SDS-PAGE and immunoblotting with anti-FLAG to detect bound Gene 33 or PAK.

Fig. 6. Selective activation of the SAPK pathway by the long form of Gene 33: Requirement for the C-terminal half of Gene 33 consisting of the 14-3-3-binding, PDZ-binding, and AH domains (the short form of Gene 33 cannot activate the SAPKs)

A, activation of MAPK pathways by Gene 33. 293 cells were transfected with HA-SAPK, ERK1, or p38 as indicated plus vector or FLAG-Gene 33. As indicated, FLAG-V12-Cdc42Hs served as a positive control for SAPK activation, FLAG-V12-Ha-Ras was a positive control for ERK1, and untagged apoptosis signal-regulating kinase-1 was a positive control for p38. HA-MAPKs were immunoprecipitated with anti-HA and assayed with GST-c-Jun (SAPK), myelin basic protein (MBP, ERK assays), or GST-activating transcription factor-2 (p38) (right panels). In the left panels, crude lysates were probed with the cognate antibodies to determine expression of the transfected constructs. B, schematic diagram of Gene 33 truncation mutants. wt, wild type; BD, binding domain; C, activation of SAPK by Gene 33 deletion constructs alone or in combination with V12-Cdc42Hs. 293 cells were cotransfected with HA-SAPK and the indicated FLAG-tagged Gene 33 and/or Cdc42Hs constructs. HA-SAPK was immunoprecipitated and assayed for GST-c-Jun kinase (top panel). Crude lysates were probed with anti-HA to detect SAPK levels (middle panel) or anti-FLAG to detect Gene 33 and Cdc42Hs levels (bottom panel). D, the short form of Gene 33 cannot activate coexpressed SAPK. The top diagram shows the structure of the long and short forms of Gene 33 (Gene 33 L and Gene 33 S, respectively). The bottom panel indicates the effect of these Gene 33 constructs on the activity of coexpressed SAPK. 293 cells were cotransfected with GST-SAPK and the indicated FLAG-tagged Gene 33 and/or V12-Cdc42Hs constructs. GST-SAPK was isolated on GSH-agarose and assayed for GST-c-Jun kinase (top panel). Crude lysates were probed with anti-GST to detect SAPK levels (middle panel) or anti-FLAG to detect Gene 33 and Cdc42Hs levels (bottom panel). IB, immunoblot.

Fig. 7. Model for the role of Gene 33 in reprogramming the cell to respond to chronic stress

Stress-induced gene 33 expression acts to maintain responses under conditions of sustained stress. The model is not intended to imply that Gene 33 represents the sole mechanism signaling hypertrophy; instead, the potential role for Gene 33 in the progression to hypertrophy is highlighted. The diagram suggests that Gene 33 might couple Cdc42 to the SAPKs under certain conditions. For this to be true, sufficient levels of Gene 33 would need to be present so as to permit appreciable binding to Cdc42. In addition, the cell would need to be treated with agonists that stimulate Cdc42 activation. Details are discussed under “Results” and “Discussion.”