Dermoscopy and reflectance confocal microscopy finding of different anatomic sites of Langerhans cell histiocytosis.

BACKGROUND: Recognizing Langerhans cell histiocytosis (LCH) might be a challenge due to its rarity. Reflectance confocal microscopy (RCM) and dermoscopy were emergent promising non-invasive technique as auxiliary tools in diagnosis of different skin conditions. However, the RCM and dermoscopic features of LCH had been less investigated. To reveal the common RCM and dermoscopic features of LCH. MATERIALS AND METHODS: Forty cases of LCH were retrospectively analyzed according to age, locations, clinical, RCM, and dermoscopic features from September 2016 to December 2022. To reveal the differences and common in clinical, RCM, and dermoscopic features that occur in different anatomic location. RESULTS: In the study, sites of predilection include the trunk 31/40 (77.5%), extremity 21/40 (52.5%), face 14/40 (35%), scalp 11/40 (27.5%), vulvar 4/40 (10%), and nail 2/40 (5%). All LCHs had the common RCM features. There were significant differences in clinical and dermoscopic features for age and lesion anatomic site. The common dermoscopic features for scalp, face, trunk, and extremity were the erythematous scaly rash, purplish-red globules or patches, scar-like streaks with ectatic vessels. While the features for nail LCH were purpuric striae, onycholysis and purulent scaly rash, and the erosive erythematous plaque and purulent scaly rash for vulvar LCH. The common RCM features of all LCH showed a focal highly reflective dense image in the surface keratin layer, epidermis architectural disarray, obscuration of dermo-epidermal junction, numerous polygonal, large, medium reflective, short dendrites cells in the epidermis, and dermis. All LCH involving the vulvar and nail did not manifest skin lesions. CONCLUSION: RCM and dermoscopy showed promising value for diagnosis and differentiation of LCH.

Cultivated land multifunctionality in undeveloped peri-urban agriculture areas in China: Implications for sustainable land management.

The spatial planning and sustainable management of peri-urban cultivated land are key aspects of national development in many countries because of the continuing expansion of urban areas and deterioration of agro-ecosystem services. Detailed geo-informational investigation of cultivated land multifunctionality and the spatial interactions and dependencies of these multiple functions is required to inform the currently weak theoretical framework of multifunctionality at the peri-urban scale. Accordingly, the objective of this study was to construct a comprehensive methodology to identify and evaluate cultivated land multifunctionality in a spatial context. Geochemical data were used to measure cultivated land multifunctionality. We evaluated two main functions-the productive function and the ecological function-of an undeveloped peri-urban agriculture (PUA) area in the northern fringe of Changchun City in the black-soil region of northeastern China. For the ecological utilization of PUA areas, tradeoff and synergy analyses of cultivated land multifunctionality and coordinated development under complex land-use patterns were measured using a bivariate local Moran's I method. Results reveal considerable spatial heterogeneity in the two functions, with hotspots or coldspots being found in the PUA area. The productive function presents a less pronounced decreasing trend along the rural-to-urban gradient compared with the ecological function. Tradeoffs and synergies between the productive function and the ecological function occur mainly in the northern (more rural) part of the PUA area, where the spatial spillover effect of urbanization is relatively low. Cultivated land functions are strongly affected by urbanization-induced land-use types, and the coordinated areas of the productive function are generally consistent with those of the ecological function. According to these results, we delineate nine zones of multifunctionality in the studied PUA area. Given the importance of harmonizing cultivated land multifunctionality to manage limited land resources in a sustainable way, application of the GIS- and geochemistry-based multifunctionality evaluation scheme proposed in this study should be used to guide peri-urban spatial planning and land-use management and inform the policy arena concerning the transition of land use in urban peripheries.

Investigation of Spatial Coupling Coordination Development: Identifying Land System States from the Adaptation-Conflict Perspective.

With the advancement of global urbanization, ecosystem conservation and sustainable land development have become major issues. In this context, the uncoordinated and imbalanced development of the land-centered human-environment system requires urgent attention, especially in rust belt cities that pose critical challenges to regional land system sustainability. Therefore, taking Changchun City from 1990 to 2020 as an example, we identified and evaluated the ecosystem service (ES) balance and land use conflict from the perspectives of internal support and external development pressure. Based on the land system adaptation and conflict results, a coupling coordination degree model (CCDM) was constructed to investigate the spatio-temporal characteristics of land system development. The results indicated that there was an obvious downward trend in the regional ES balance, while areas with higher ES balance were mainly distributed in the eastern ecologically sound areas, and southern built-up areas presented deficient ES balance levels (i.e., demand exceeding supply), with a significant expansion trend from 1990 to 2020. Land use conflict was especially prominent in areas experiencing rapid rural-urban transformation, and the hot spots expanded noticeably. The spatio-temporal differences in the coupling coordination degree of ES balance and land use conflict were significant, whereas the land system of the study area has always been dominated by a balanced development pattern throughout the research period, except for the urban center, which tended to be in a stage of uncoordinated development, with the ES balance blocked. These findings suggest that it is necessary to coordinate urban and adjacent regions through regionally integrated efforts to alleviate the ES imbalance. This research can provide a scientific reference for analyzing regional land system states, coordinating the sustainable spatial development of ecosystems, and implementing revitalization strategies to achieve win-win land system goals.

[Risk Zoning of Heavy Metals in a Peri-urban Area in the Black Soil Farmland Based on Agricultural Products].

Agricultural products are a primary pathway for humans to accumulate heavy metals (HMs) via the soil-crop system and should therefore should be included as a crucial part of the food security in our country. Given that previous studies on protection zoning for preventing farmland HM pollution rarely considered agricultural products as a basic element, this study attempted to establish a zoning system for farmland HM prevention, which was based on the perspective of agricultural product pollution. We subsequently took a representative peri-urban area in the black soil region, which was provided with a higher risk of being polluted, as an empirical case. The results indicated that:① the comprehensive quality index of agricultural products (IICQAP) was 1.09, illustrating only a mild HM pollution, with Pb and Ni having the highest accumulation levels; ② the human health risk index (QHI) was 0.61, showing no risk for human health; and ③ the designed zoning method revealed 89.45% of the farmlands to be risk-free at the moment and 10.55% of the farmlands to be under low risk of HM pollution in agricultural products. According to the zoning results, we suggested prioritized protection and an early-warning strategy, respectively, and further recommended prevention methods such as accumulation intervention, crop restructuring, and in-situ passivation. The results served to enrich the theoretical basis for preventing farmland HM pollution, to reinforce the management standards for agricultural products in the black soil region, and also to build a differentiated urban-rural farmland protection system.

Inhibition of thromboxane a synthase activity enhances steroidogenesis and steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells.

The cyclooxygenase-2 (COX2)-dependent inhibition of Leydig cell steroidogenesis has been demonstrated. To understand the mechanism for this effect of COX2, the present study examined the role of an enzyme downstream of COX2, namely thromboxane A synthase (TBXAS), in steroidogenesis. Inhibition of TBXAS activity with the inhibitor furegrelate induced a concentration-dependent increase in cAMP-induced steroidogenic acute regulatory (StAR) protein in MA-10 mouse Leydig cells. The increase in StAR protein occurred concomitantly with a significant increase in steroid hormone production. Similar results were obtained in StAR promoter activity assays and RT-PCR analyses of StAR mRNA levels, suggesting that inhibition of TBXAS activity enhanced StAR gene transcription. These observations were corroborated when TBXAS expression was specifically inhibited by RNA interference. Although the RNA interference reduced mRNA levels of TBXAS, it increased StAR mRNA levels, StAR protein, and steroidogenesis. Additional studies indicated that inhibition of TBXAS activity reduced DAX-1 protein, a repressor in StAR gene transcription. In the absence of cAMP, inhibition of TBXAS activity did not induce a significant increase in steroid hormone and StAR protein. However, addition of a low level of cAMP analogs dramatically increased steroidogenesis. Lastly, inhibition of protein kinase A activity essentially abolished the steroidogenic effect of the TBXAS inhibitor. Thus, the results from the present study suggest that a minimal level of protein kinase A activity is required for the steroidogenic effect of the TBXAS inhibitor and that inhibition of TBXAS activity or its expression increase the steroidogenic sensitivity of MA-10 mouse Leydig cells to cAMP stimulation.

The involvement of epoxygenase metabolites of arachidonic acid in cAMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression.

The essential role of arachidonic acid (AA) in steroidogenesis has been previously demonstrated. The present study continues the investigation into how AA regulates steroidogenesis by examining the effects of epoxygenase-derived AA metabolites on cAMP-stimulated steroidogenic acute regulatory (StAR) gene expression and steroid hormone production in MA-10 mouse Leydig cells. The HPLC analysis of cell extracts from MA-10 cells treated with the cAMP analog dibutyryl cAMP (dbcAMP) demonstrated an increase in three epoxygenase-generated AA metabolites: 5,6-epoxyeicosatrienoic acid (EET), 8,9-EET, and 11,12-EET. Incubating MA-10 cells with each of the EETs induced a dose-dependent increase in StAR protein expression and steroid hormone production in the presence of dbcAMP. These metabolites also significantly enhanced StAR gene transcription as determined by luciferase assays of StAR promoter activity and reverse transcriptase-PCR analysis of StAR mRNA levels. While the EETs enhanced steroidogenesis, inhibiting the activity of protein kinase A (PKA) abolished the stimulatory effects of these AA metabolites on StAR expression and steroid hormone production. This study suggests that cAMP stimulation of MA-10 cells increases epoxygenase-generated AA metabolites and the co-action of these metabolites with PKA significantly increases StAR gene expression and steroid hormone production.

Multiple signaling pathways regulating steroidogenesis and steroidogenic acute regulatory protein expression: more complicated than we thought.

Steroid hormone biosynthesis in steroidogenic cells is regulated through trophic hormone activation of protein kinase A (PKA) signaling pathways. However, many examples of the regulation of steroid synthesis via pathways other than the PKA pathway have been documented. In some cases these pathways act independently of PKA activation whereas in other cases, they act synergistically with it. The current understanding of additional signaling pathways and factors, such as the protein kinase C pathway, arachidonic acid metabolites, growth factors, chloride ion, the calcium messenger system, and others capable of regulating/modulating steroid hormone biosynthesis, and in many cases steroidogenic acute regulatory protein expression, are discussed in this review.

Cyclooxygenase-2 regulation of the age-related decline in testosterone biosynthesis.

The age-related decline in testosterone biosynthesis in testicular Leydig cells has been well documented, but the mechanisms involved in the decline are not clear. Recent studies have described a cyclooxygenase-2 (COX2)-dependent tonic inhibition of Leydig cell steroidogenesis and expression of the steroidogenic acute regulatory protein (StAR). The present study was conducted to determine whether COX2 protein increases with age in rat Leydig cells and whether COX2 plays a role in the age-related decline in testosterone biosynthesis. Our results indicate that from 3 months of age to 30 months, COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells, StAR protein decreased to 33%, and blood testosterone concentration and testosterone biosynthesis in Leydig cells decreased to 41 and 33%, respectively. Further experiments demonstrated that overexpressing COX2 in MA-10 mouse Leydig cells inhibited StAR gene expression and steroidogenesis and that the inhibitory effects of COX2 could be reversed by blocking COX2 activity. Notably, incubation of aged Leydig cells with the COX2 inhibitor NS398 enhanced their testosterone biosynthesis. Blood testosterone concentrations in aged rats fed the COX2 inhibitor DFU, at doses of 5, 10, 15, and 20 mg/kg body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in the rats receiving no DFU. The present study suggests a novel mechanism in male aging involving COX2 and a potential application of the mechanism to delay the age-related decline in testosterone biosynthesis.

The decline in testosterone biosynthesis during male aging: a consequence of multiple alterations.

The decline in blood testosterone concentration during the course of male aging results in decreases in many physiological functions. However, the mechanism(s) responsible for this decline is not clear. Previous observations have suggested the involvement of multiple alterations or defects that inhibit the activities of proteins involved in steroidogenesis and result in reduced testosterone biosynthesis. Recent studies have demonstrated an age-related increase in cyclooxygenase-2 (COX2) activity and its tonic inhibition of steroidogenic acute regulatory gene expression and steroidogenesis in rat Leydig cells. These findings indicate the presence of a novel mechanism in male aging involving COX2 and suggest the potential application of COX2 inhibitors or other interventions in this mechanism to delay the decline in testosterone biosynthesis in aged males.

Inhibition of cyclooxygenase-2 activity enhances steroidogenesis and steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells.

To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mM dibutyryl cAMP (Bt(2)cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 microM of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.7-fold and 34.3-fold, respectively. In the presence of indomethacin, the level of Bt(2)cAMP required for maximal steroidogenesis was reduced from 1.0 mM to 0.25 mM. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression, suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. Although SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt(2)cAMP-stimulated StAR protein expression and steroid production. Overexpression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.

Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression.

The rate-limiting, committed, and regulatable step in steroid hormone biosynthesis is the transport of cholesterol from the outer to the inner mitochondrial membrane, a process that is mediated by the steroidogenic acute regulatory (StAR) protein. In steroidogenic cells, the StAR protein is regulated by cAMP-dependent mechanisms. However, the StAR promoter lacks a consensus cAMP response-element (CRE), suggesting the involvement of alternate regulatory factor(s) in cAMP responsiveness. These regulatory elements are found to be located in a transcription factor-binding site-rich region (consisting of approximately 150 nucleotides upstream of the transcription start site) of the StAR promoter, and appears to be the most important region in regulating transcription of the StAR gene. The StAR promoter sequences in mouse, rat and human are highly homologous, and in the absence of a canonical CRE, multiple cis-elements have been shown to be instrumental in the regulation of StAR gene expression. Nevertheless, it has become apparent that functional cooperation, interaction, and alteration of different transcription factors are involved in the fine-tuning of the regulatory events associated with StAR gene transcription.

Effects of apigenin on steroidogenesis and steroidogenic acute regulatory gene expression in mouse Leydig cells.

Previous studies reported that the age-related decline in testosterone biosynthesis is associated with a decrease in the steroidogenic acute regulatory (StAR) protein which regulates the rate-limiting step of testosterone biosynthesis. To explore the possibility of delaying this decline using a dietary approach, we have examined the effect of a natural flavonoid, apigenin, on StAR gene expression in mouse Leydig cells. Incubation of these cells with the flavonoid enhanced cyclic adenosine monophosphate (cAMP)-induced steroidogenesis and StAR protein expression. The results from the analyses of StAR mRNA by reverse transcription-polymerase chain reaction and the luciferase assays of StAR promoter activity indicated that this flavonoid enhanced StAR gene expression at the level of transcription. Further studies showed that apigenin blocked the thromboxane A2 receptor and interrupted the signaling through the cyclooxygenase-2-thromboxane A synthase-thromboxane A2-receptor pathway, resulting in a reduction of DAX-1 (dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene-1) protein, a transcriptional repressor of StAR gene expression. When DAX-1 protein was reduced, the sensitivity of the Leydig cells was dramatically enhanced, with sub-threshold level of cAMP being able to induce maximal levels of StAR protein expression and steroid hormone production. The present study suggests a potential application of apigenin to improve StAR protein expression and steroidogenic sensitivity of aging Leydig cells.

Blocking L-type calcium channels reduced the threshold of cAMP-induced steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells.

Previous studies have reported the roles of Ca(2+) in steroidogenesis. The present study has investigated an inhibitory effect of Ca(2+) influx through L-type Ca(2+) channels on gene expression of steroidogenic acute regulatory (STAR) protein that regulates the transfer of substrate cholesterol to the inner mitochondrial membrane for steroidogenesis. Blocking Ca(2+) influx through L-type Ca(2+) channels using the selective Ca(2+) channel blocker, nifedipine, markedly enhanced cAMP-induced STAR protein expression and progesterone production in MA-10 mouse Leydig cells. This was confirmed by utilization of different L-type Ca(2+) channel blockers. Reverse transcription-PCR analyses of Star mRNA and luciferase assays of Star promoter activity indicated that blocking Ca(2+) influx through L-type Ca(2+) channels acted at the level of Star gene transcription. Further studies showed that blocking the Ca(2+) channel enhanced Star gene transcription by depressing the expression of DAX-1 (NR0B1 as listed in the MGI Database) protein, a transcriptional repressor of Star gene expression. It was also observed that there is a synergistic interaction between nifedipine and cAMP. Normally, sub-threshold levels of cAMP are unable to induce steroidogenesis, but in the presence of the L-type Ca(2+) channel blocker, they increased STAR protein and steroid hormone to the maximal levels. However, in the absence of minimal levels of cAMP, none of the L-type Ca(2+) channel blockers are able to induce Star gene expression. These observations indicate that Ca(2+) influx through L-type Ca(2+) channels is involved in an inhibitory effect on Star gene expression. Blocking L-type Ca(2+) channel attenuated the inhibition and reduced the threshold of cAMP-induced Star gene expression in Leydig cells.

Involvement of the thromboxane A2 receptor in the regulation of steroidogenic acute regulatory gene expression in murine Leydig cells.

Recent studies suggested an involvement of thromboxane A2 in cyclooxygenase-2-dependent inhibition of steroidogenic acute regulatory (StAR) gene expression. The present study further investigated the role of thromboxane A2 receptor in StAR gene expression and steroidogenesis in testicular Leydig cells. The thromboxane A2 receptor was detected in several Leydig cell lines. Blocking thromboxane A2 binding to the receptor using specific antagonist SQ29548 or BM567 resulted in dose-dependent increases in StAR protein and steroid production in MA-10 mouse Leydig cells. The results were confirmed with Leydig cells isolated from rats. StAR promoter activity and StAR mRNA level in the cells were also increased after the treatments, suggesting an involvement of the thromboxane A2 receptor in StAR gene transcription. Furthermore study indicated that blocking the thromboxane A2 receptor reduced dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 protein, a transcriptional repressor of StAR gene expression. Specific binding of the antagonists to the receptors on cellular membrane was demonstrated by binding assays using (3)H-SQ29548 and binding competition between (3)H-SQ29548 and BM567. Whereas SQ29548 enhanced cAMP-induced StAR gene expression, in the absence of cAMP, it was unable to increase StAR protein and steroidogenesis. However, when the receptor was blocked by the antagonist, subthreshold levels of cAMP were able to induce maximal levels of StAR protein expression, suggesting that blocking the thromboxane A2 receptor increase sensitivity of MA-10 cells to cAMP stimulation. Taken together, the results from the present and previous studies suggest an autocrine loop, involving cyclooxygenase-2, thromboxane A synthase, and thromboxane A2 and its receptor, in cyclooxygenase-2-dependent inhibition of StAR gene expression.

Chrysin, a natural flavonoid enhances steroidogenesis and steroidogenic acute regulatory protein gene expression in mouse Leydig cells.

During the aging process of males, testosterone biosynthesis declines in testicular Leydig cells resulting in decreases in various physiological functions. To explore the possibility of delaying the decline using food supplements, we have studied steroidogenic effects of a natural flavonoid, chrysin, in mouse Leydig cells. Chrysin dramatically increased cyclic AMP (cAMP)-induced steroidogenesis in MA-10 mouse Leydig tumor cells. This result was confirmed using Leydig cells isolated from mouse testes. The steroidogenic effect of chrysin is not associated with an increase in expression of the P450 side-chain cleavage enzyme, required for the conversion of cholesterol to pregnenolone. In addition, when 22(R)hydroxylcholesterol was used as a substrate, chrysin induced a non-significant increase in steroid hormone, suggesting that the majority of the observed increase in steroidogenesis was due to the increased supply of substrate cholesterol. These observations were corroborated by showing that chrysin induced a marked increase in the expression of steroidogenic acute regulatory (StAR) protein, the factor that controls mitochondrial cholesterol transfer. Also, chrysin significantly increased StAR promoter activity and StAR mRNA level. Further studies indicated that this compound depressed expression of DAX-1, a repressor in StAR gene transcription. In the absence of cAMP, chrysin did not increase steroidogenesis. However, when a sub-threshold level of cAMP was used, StAR protein and steroid hormone were increased by chrysin to the levels seen with maximal stimulation of cAMP. These results suggest that while chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.

Short-term supplementation of COX-2 inhibitor suppresses bone turnover in gonad-intact middle-aged male rats.

There is an increasing body of evidence supporting the idea that nonsteroidal antiinflammatory drugs can effectively suppress ovariectomy-induced bone loss in adult rats. The present study investigated the effects of supplementation of selective cyclooxygenase-2 inhibitor [5,5-dimethyl-3-(3 flurophenyl)-4-(4 methylsulphonal) phenyl-2 (6H) furanone, DFU] to diets on bone metabolism, bone mineral density (BMD), and histomorphometry in middle-aged male rats. Forty 16-month-old male rats (n = 8/group) were fed diets containing 0 (control), 5, 10, 15, or 20 mg/kg DFU body weight/day for 4 weeks. Supplementation of DFU resulted in higher plasma levels of pyridinoline, insulin-like growth factor I, and parathyroid hormone, but lower bone-specific alkaline phosphatase activity, prostaglandin E(2,) and 1,25-(OH)(2) vitamin D(3). Compared to the control, DFU supplementation at 20 mg/kg did not affect total scanned area, BMD, and bone mineral content as determined by DEXA. Histomorphometric data showed that rats fed DFU at 20 mg/kg had lower values of mineralizing surface, eroded surface, mineral apposition rate, and bone formation rate, but no difference in trabecular thickness, number, and separation compared to the control. The present data demonstrate that the short-term supplementation of selective COX-2 inhibitor to gonad-intact middle-aged male rats resulted in suppression of bone formation and increased bone resorption, and thereby suppressed bone turnover rate, without significant loss of bone. Such action was through modulating local factors and systemic hormones.

Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries.

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.

Mechanisms of epidermal growth factor signaling: regulation of steroid biosynthesis and the steroidogenic acute regulatory protein in mouse Leydig tumor cells.

Steroid hormone biosynthesis in the adrenals and gonads is regulated by the steroidogenic acute regulatory (StAR) protein through its action in mediating the intramitochondrial transport of cholesterol. A role for epidermal growth factor (EGF) in modulating steroidogenesis has been previously determined, but the mechanism of its action remains unknown. The present investigation was designed to explore the potential mechanism of action of mouse EGF (mEGF) in the regulation of steroid biosynthesis and StAR protein expression in mLTC-1 mouse Leydig tumor cells. We show that treatment of mLTC-1 cells with mEGF significantly increased the levels of progesterone (P), StAR protein, and StAR mRNA in a time- and dose-dependent manner. The coordinate induction of P synthesis and StAR gene expression by mEGF was effectively inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. Also, longer exposure of mLTC-1 cells to mEGF produced a marked decrease in LH-receptor mRNA expression. These effects of mEGF were exerted through high-affinity binding sites (K(d) approximately 0.53 nmol/L) in these cells. It was also determined that the arachidonic acid (especially lipoxygenase metabolites) and mitogen-activated protein kinase pathways were also involved in the mEGF-induced steroidogenic response. However, involvement of the latter pathway was further assessed in nonsteroidogenic COS-1 cells transfected with the Elk1 trans-reporting plasmids and resulted in a significant increase in luciferase activity in response to mEGF. Furthermore, deletion and mutational analyses demonstrated a predominant involvement of activator protein-1 in addition to the multiple mEGF responsive elements found within the 5'-flanking region (-151/-1 base pairs) of the mouse StAR gene. These findings provide novel insights into the mEGF-induced regulatory cascades associated with steroid synthesis and StAR protein expression in mouse Leydig cells.