The ERK Signaling Cascade Inhibits Gonadotropin-Stimulated Steroidogenesis.

The response of granulosa cells to Luteinizing Hormone (LH) and Follicle- Stimulating Hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the Steroidogenic Acute Regulatory Protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

Nonassembled human chorionic gonadotropin subunits and alphaalpha-homodimers use fast-track processing in the secretory pathway in contrast to alphabeta-heterodimers.

In multimeric glycoproteins, like glycoprotein hormones, mutual subunit interactions are required for correct folding, assembly, and transport in the secretory pathway. However, character and time course of these interactions need further elucidation. The influence of the glycoprotein hormone alpha-subunit (GPHalpha) on the folding of the human chorionic gonadotropin (hCG) beta-subunit (hCGbeta) in hCG alphabeta-heterodimers was investigated in [(35)S]Met/Cys-labeled JEG-3 cells. Completeness of disulfide bridge formation during the time course of folding was estimated by labeling with [(3)H]N-ethylmaleinimide of free thiol groups not yet consumed. Subunit association took place between immature hCGbeta (high (3)H/(35)S ratio) and almost completely folded GPHalpha. Analysis revealed a highly dynamic maturation process comprising of at least eight main hCGbeta folding intermediates (molecular masses from 107 to 28 kDa) that could be micro-preparatively isolated and characterized. These hCGbeta variants developed while being associated with GPHalpha. The 107-kDa variant was identified as a complex with calnexin. In contrast to hCG alphabeta-heterodimers, free nonassociated hCGbeta, free large GPHalpha, and GPHalphaalpha homodimers showed a fast-track-like processing in the secretory pathway. At 10 min before hCG secretion, sialylation of these variants had already been completed in the late Golgi, whereas hCG alphabeta-heterodimers had still not arrived medial Golgi. This shows that the GPHalpha in the hCG alphabeta-heterodimers decelerates the maturation of the hCGbeta portion in the heterodimer complex. This results in a postponed approval of hCG alphabeta-heterodimers by the endoplasmic reticulum quality control unlike GPHalphaalpha homodimers, free hCGbeta, and GPHalpha subunits.

Rapid maturation of glycoprotein hormone free alpha-subunit (GPHalpha) and GPHalpha alpha homodimers.

The dynamics of glycoprotein hormone alpha-subunit (GPHalpha) maturation and GPHalpha alpha homodimer formation were studied in presence (JEG-3 choriocarcinoma cells) and absence (HeLa cells) of hCGbeta. In both cases, the major initially occurring GPHalpha variant in [35S]Met/Cys-labeled cells carried two N-glycans (M(r app) = 22 kDa). Moreover, a mono-N-glycosylated in vivo association-incompetent GPHalpha variant (M(r app) = 18 kDa) was observed. In JEG-3 cells the early 22-kDa GPHalpha either associated with hCGbeta, or showed self-association to yield GPHalpha alpha homodimers, or was later converted into heavily glycosylated large free GPHalpha (M(r app) = 24 kDa). Micro-preparative isolation of intracellular GPHalpha alpha homodimers of JEG-3 cells and their conversion by reduction revealed that they consisted of 22-kDa GPHalpha monomers and not of large free GPHalpha. In HeLa cells, the large free GPHalpha variant was not observed, whereas GPHalpha alpha homodimers were present. Intracellularly, early GPHalpha alpha homodimers (35 kDa) and late variants (JEG-3: 44 kDa, HeLa: 39 kDa) were found. Both cell types secreted 45 kDa GPHalpha alpha homodimers. Large free GPHalpha and GPHalpha alpha homodimers were more rapidly sialylated than hCG alphabeta-heterodimers indicating a sequestration mechanism in the secretory pathway. In GPHalpha alpha homo- as well as hCG alphabeta-heterodimers the subunit interaction site, located on loop 2 of GPHalpha (amino acids 33-42), became immunologically inaccessible indicating similar spatial orientation of GPHalpha in both types of dimers. The studies demonstrate the formation, in vivo dynamics of GPHalpha alpha homodimers, and the pathways of the cellular metabolism of variants of GPHalpha, monoglycosylated GPHalpha and large free GPHalpha.

Time-dependent folding of immunological epitopes of the human chorionic gonadotropin beta-subunit.

We have explored the possibility to use 14 different monoclonal antibodies in order to follow the formation of the respective epitopes during the biosynthesis of hCG subunits and their association in JEG-3 choriocarcinoma cells using pulse (30s to 5 min)-chase (0-180 min) experiments. We found central cystine knot epitope structures (epitope beta1) to be formed immediately and simultaneously with epitopes on the protruding hCG-beta loops 1 and 3. We found also differences in the time-dependent folding of beta2 and beta4 epitopes, which are highly overlapping structures on the loops 1+3. These differences were reinforced by decreasing the temperature during the pulse-chase experiments to 25 degrees C. Moreover, we describe for the first time an intracellular intact hCG beta-subunit form that showed the transient expression of the hCG-beta-core fragment epitope beta11 in the course of the maturation of this subunit which casts new light on the presence of hCG-beta-core fragment in Down's syndrome, tumors and pregnancy.

Activation of tubular epithelial cells in diabetic nephropathy.

Previous studies have shown that renal function in type 2 diabetes correlates better with tubular changes than with glomerular pathology. Since advanced glycation end products (AGEs; AGE-albumin) and in particular carboxymethyllysine (CML) are known to play a central role in diabetic nephropathy, we studied the activation of nuclear factor kappaB (NF-kappaB) in tubular epithelial cells in vivo and in vitro by AGE-albumin and CML. Urine samples from healthy control subjects (n = 50) and type 2 diabetic patients (n = 100) were collected and tested for excretion of CML and the presence of proximal tubular epithelial cells (pTECs). CML excretion was significantly higher in diabetic patients than in healthy control subjects (P < 0.0001) and correlated with the degree of albuminuria (r = 0.7, P < 0.0001), while there was no correlation between CML excretion and HbA(1c) (r = 0.03, P = 0.76). Urine sediments from 20 of 100 patients contained pTECs, evidenced by cytokeratin 18 positivity, while healthy control subjects (n = 50) showed none (P < 0.0001). Activated NF-kappaB could be detected in the nuclear region of excreted pTECs in 8 of 20 patients with pTECs in the urine sediment (40%). Five of eight NF-kappaBp65 antigen-positive cells stained positive for interleukin-6 (IL-6) antigen (62%), while only one of the NF-kappaB-negative cells showed IL-6 positivity. pTECs in the urine sediment correlated positively with albuminuria (r = 0.57, P < 0.0001) and CML excretion (r = 0.55, P < 0.0001). Immunohistochemistry in diabetic rat kidneys and a human diabetic kidney confirmed strong expression of NF-kappaB in tubular cells. To further prove an AGE/CML-induced NF-kappaB activation in pTECs, NF-kappaB activation was studied in cultured human pTECs by electrophoretic mobility shift assays (EMSAs) and Western blot. Stimulation of NF-kappaB binding activity was dose dependent and was one-half maximal at 250 nmol/l AGE-albumin or CML and time dependent at a maximum of activation after 4 days. Functional relevance of the observed NF-kappaB activation was demonstrated in pTECs transfected with a NF-kappaB-driven luciferase reporter plasmid and was associated with an increased release of IL-6 into the supernatant. The AGE- and CML-dependent activation of NF-kappaBp65 and NF-kappaB-dependent IL-6 expression could be inhibited using the soluble form of the receptor for AGEs (RAGE) (soluble RAGE [sRAGE]), RAGE-specific antibody, or the antioxidant thioctic acid. In addition transcriptional activity and IL-6 release from transfected cells could be inhibited by overexpression of the NF-kappaB-specific inhibitor kappaBalpha. The findings that excreted pTECs demonstrate activated NF-kappaB and IL-6 antigen and that AGE-albumin and CML lead to a perpetuated activation of NF-kappaB in vitro infer that a perpetuated increase in proinflammtory gene products, such as IL-6, plays a role in damaging the renal tubule.