Antigen Concentrations as an Indicator of Clinical Remission and Disease Relapse in Cats with Histoplasmosis.

BACKGROUND: Treatment monitoring is subjective and disease relapse is common in cats with histoplasmosis. The Histoplasma antigen enzyme immunoassay (EIA) is a noninvasive test used for determining disease remission and detecting disease relapse in humans with histoplasmosis. The utility of the antigen EIA for these purposes in cats remains unknown. HYPOTHESIS/OBJECTIVES: Those Histoplasma antigen concentrations in urine and serum would decline with antifungal treatment and that antigen elimination would be an indicator of clinical remission in cats with histoplasmosis treated with antifungal treatment. ANIMALS: Fifteen client-owned cats with histoplasmosis. METHODS: Masked observational study. Cats were monitored monthly during antifungal treatment. Time of clinical remission and serum and urine antigen elimination were determined for each cat. RESULTS: Twelve of 15 cats achieved clinical remission. At the time of diagnosis, antigen was detectable in urine in 14/15 (93%) cats and in serum in 11/15 (73%) cats. Both serum (P < .0005) and urine (P < .0001) antigen concentrations significantly decreased over time with effective treatment. Antigen elimination was sensitive [urine, 90.0% (95% CI 72.3-97.4%); serum, 90.4% (68.2-98.3%)] but less specific [urine, 64.6% (51.7-75.8%); serum, 52.1% (37.4-66.5%)] for disease remission. Urine antigen was positive in both cats and serum antigen was positive in 1 cat at the time of disease relapse. CONCLUSIONS AND CLINICAL IMPORTANCE: Measurement of Histoplasma antigen in urine and serum might be useful tests for determining disease remission and relapse in cats with histoplasmosis. Further research is needed to investigate the importance of low-level antigenemia and antigenuria.

Experimental infection of domestic cats (Felis domesticus) with Cytauxzoon manul from Pallas' cats (Otocolobus manul).

In a random, blind study, six domestic cats were assigned to two treatment groups that received either sterile water or dexamethasone by subcutaneous injection prior to intravenous inoculation with Pallas' cat (Otocolobus manul) blood infected with Cytauxzoon manul. A seventh domestic cat served as a control and was inoculated only with sterile water. Cats were monitored for clinical signs consistent with cytauxzoonosis, and periodically screened for hemoparasitemia. All domestic cats (6/6) that received Pallas' cat blood infected with C. manul developed a low but detectible parasitemia by 9 days post-inoculation, yet remained clinically healthy. All domestic cats (7/7) were subsequently challenged with Cytauxzoon felis and developed clinical signs typical of cytauxzoonosis within 5 days post-challenge. Affected animals were euthanized and cytauxzoonosis was confirmed by histopathology. While inoculation of domestic cats with Pallas' cat blood infected with C. manul induced a parasitemia, it did not cause disease or provide protection against challenge with C. felis. Further studies are warranted to determine the potential for interspecies transmission and disease with C. manul.

Enhanced sensitivity to apoptosis in Ras-transformed thyroid cells.

Ras mutations occur at high frequency in thyroid cancer. In vitro, the effects of Ras in thyroid cells are pleiotropic in that expression of activated Ras has been reported to stimulate proliferation and apoptosis. An understanding of the factors that contribute to the survival versus demise of Ras-transformed cells is essential to our understanding of the contribution of Ras to thyroid neoplasia and other cancers. Constitutive expression of oncogenic H-Ras sensitized Wistar rat thyroid (WRT) cells to apoptosis stimulated by multiple insults. When deprived of matrix attachment, Ras-transformed cells perished by apoptotic cell death at a high frequency. In contrast, parental cells were more resistant to suspension-induced cell death. Ras effects on anchorage-independent cell death were reproduced by a mutant protein that signals selectively to Raf-1, but not by mutant Ras that preferentially binds to RalGDS. Expression of a Ras mutant that selectively activates PI3K resulted in substantial protection from detachment-induced cell death. MAPK activity was increased in adherent Ras12V- and Ras12V35S-expressing cells, but abolished upon detachment. Interestingly, impaired MAPK activity was sufficient to stimulate apoptosis in adherent Ras-transformed cells, but not in parental cells. Treatment with a PI3K inhibitor also stimulated apoptosis selectively in Ras-transformed cells. These results demonstrate that constitutive expression of activated Ras elicits differential effects on the survival of thyroid cells. Moreover, Ras expression results in a greater dependence of thyroid cells on MAPK and PI3K activity for their survival.

A genotypically unique Babesia gibsoni-like parasite recovered from a dog in Oklahoma.

A small Babesia gibsoni-like parasite was identified and isolated as the cause of clinical babesiosis in a dog from Oklahoma. Because this was potentially the first documented case of B. gibsoni infection in Oklahoma, further characterization was warranted, and the 18S nuclear small subunit ribosomal RNA gene was sequenced. Sequence comparison with other piroplasms from dogs showed significant nucleotide sequence differences between this isolate and both B. canis and B. gibsoni. These findings demonstrate that in domestic dogs in North America there are at least 2 "small" B. gibsoni-like organisms with distinct nucleotide sequences and that the geographic distribution of the "small" canine Babesia species may be wider than previously recognized.

Coordinated regulation of Rap1 and thyroid differentiation by cyclic AMP and protein kinase A.

Originally identified as an antagonist of Ras action, Rap1 exhibits many Ras-independent effects, including a role in signaling pathways initiated by cyclic AMP (cAMP). Since cAMP is a critical mediator of the effects of thyrotropin (TSH) on cell proliferation and differentiation, we examined the regulation of Rap1 by TSH in a continuous line of rat thyroid-like cells. Both cAMP and protein kinase A (PKA) contribute to the regulation of Rap1 activity and signaling by TSH. TSH activates Rap1 through a cAMP-mediated and PKA-independent mechanism. TSH phosphorylates Rap1 in a PKA-dependent manner. Interference with PKA activity blocked phosphorylation but not the activation of Rap1. Rather, PKA inhibitors prolonged Rap1 activation, as did expression of a Rap1A mutant lacking a PKA phosphorylation site. These results indicate that PKA elicits negative feedback regulation on cAMP-stimulated Rap1 activity in some cells. The dual regulation of Rap1 by cAMP and PKA extends to downstream effectors. The ability of TSH to stimulate Akt phosphorylation was markedly enhanced by the expression of activated Rap1A and was repressed in cells expressing a putative dominant-negative Rap1A mutant. Although the expression of activated Rap1A was sufficient to stimulate wortmannin-sensitive Akt phosphorylation, TSH further increased Akt phosphorylation in a phosphatidylinositol 3-kinase- and PKA-dependent manner. The ability of TSH to phosphorylate Akt was impaired in cells expressing a Rap1A mutant that could be activated but not phosphorylated. These findings indicate that dual signals, Rap1 activation and phosphorylation, contribute to TSH-stimulated Akt phosphorylation. Rap1 plays an essential role in cAMP-regulated differentiation. TSH effects on thyroid-specific gene expression, but not its effects on proliferation, were markedly enhanced in cells expressing activated Rap1A and repressed in cells expressing a dominant-negative Rap1A mutant. These findings reveal complex regulation of Rap1 by cAMP including PKA-independent activation and PKA-dependent negative feedback regulation. Both signals appear to be required for TSH signaling to Akt.

Akt regulates cell survival and apoptosis at a postmitochondrial level.

Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. We previously showed that transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Here we show that stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c induced caspase activation in a cell-free assay while lysates from Bcl-2-expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c-induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9.

Quantification of hepatic and renal cortical echogenicity in clinically normal cats.

OBJECTIVE: To quantitatively determine echogenicity of the liver and renal cortex in clinically normal cats. ANIMALS: 17 clinically normal adult cats. PROCEDURE: 3 ultrasonographic images of the liver and the right kidney were digitized from video output from each cat. Without changing the ultrasound machine settings, an image of a tissue-equivalent phantom was digitized. Biopsy specimens of the right renal cortex and liver were obtained for histologic examination. Mean pixel intensities within the region of interest (ROI) on hepatic, renal cortical, and tissue-equivalent phantom ultrasonographic images were determined by histogram analysis. From ultrasonographic images, mean pixel intensities for hepatic and renal cortical ROI were standardized by dividing each mean value by the mean pixel intensity from the tissue-equivalent phantom. RESULTS: The mean (+/- SD) standardized hepatic echogenicity value was 1.06 +/- 0.02 (95% confidence interval, 1.02 to 1.10). The mean standardized right renal cortical echogenicity value was 1.04 +/- 0.02 (95% confidence interval, 1.01 to 1.08). The mean combined standardized hepatic and renal cortical echogenicity value was 1.02 +/- 0.05 (95% confidence interval, 0.99 to 1.04). CONCLUSIONS AND CLINICAL RELEVANCE: Quantitative determination of hepatic and renal cortical echogenicity in cats is feasible, using histogram analysis, and may be useful for early detection of diffuse parenchymal disease and for serially evaluating disease progression.

Cats surviving natural infection with Cytauxzoon felis: 18 cases (1997-1998).

Eighteen cats surviving natural infection with Cytauxzoon felis were identified. All cats came from a limited geographic area in northwestern Arkansas and northeastern Oklahoma. Clinical signs in most cats were similar to those described for cytauxzoonosis; however, 4 cats were asymptomatic. All cases were initially diagnosed by microscopic identification of signet ring-shaped piroplasms in erythrocytes of peripheral blood smears. Four of 4 cats tested had detectable serum antibodies to C felis. Four different cats were positive by polymerase chain reaction (PCR). Partial sequencing of the PCR product from 1 cat revealed >99% homology with the reported sequence of C felis. Repeated examination of blood smears from 12 cats revealed that the erythroparasitemia was generally persistent for the duration of follow-up (3-154 days). Survival did not seem dependent on treatment, as only 1 cat was treated with a drug with potential antiprotozoal activity (imidocarb dipropionate), and 4 cats received no treatment. The findings of this study may indicate the existence of a less virulent strain of C felis.

Cyclic AMP activates Ras.

In addition to protein kinase A (PKA), cAMP regulates the activity of cAMP-gated channels and Rap1-specific guanine nucleotide exchange factors. We tested the hypothesis that the targets of cAMP might also include regulators of the Ras protooncogene. In rat thyroid cells, thyrotropin (TSH) stimulates proliferation through a cAMP-mediated pathway that requires Ras activity. Interference with Ras impairs DNA synthesis stimulated by TSH as well as cAMP elevating agents and analogs, demonstrating that the requirement for Ras lies down-stream of cAMP. Although cAMP stimulates proliferation, microinjection of the purified PKA catalytic subunit failed to do so, suggesting that factors in addition to PKA are required for cAMP-stimulated cell cycle progression. When added to thyroid cells expressing human Ha-Ras, TSH rapidly and markedly increased the proportion of GTP-bound Ras. Ras activity was increased within 1 min of TSH addition, maximal at 5-15 min, and declined to basal levels 30-60 min after hormone treatment. Cyclic AMP elevating agents elicited similar effects on Ras, indicating that TSH activates Ras through a cAMP-mediated pathway. Although cAMP-mediated, Ras activation by TSH and cAMP was independent of PKA activity. Moreover, cAMP-stimulated Ras activation was not impaired by tyrosine kinase inhibitors. These results indicate that cAMP activates targets in addition to PKA in thyroid cells, and that these targets may include regulators of Ras. The ability of cAMP elevating agents to activate Ras in addition to PKA may explain the inability of the PKA catalytic subunit to stimulate DNA synthesis in thyroid cells.

Cadherin repertoire determines partner-specific gap junctional communication during melanoma progression.

Reduced gap junction activity has long been implicated in tumorigenesis. To elucidate the potential role of intercellular communication in melanoma development, we examined gap junctional capability of melanocytic cells from various stages of tumor progression in coculture models using dye transfer assays. Normal melanocytes coupled with keratinocytes by gap junctional formation, whereas melanoma cells did not. Instead, melanoma cells communicated among themselves and with fibroblasts. This switch in communication partners coincided with a shift from E-cadherin to N-cadherin expression during melanoma development. Forced expression of E-cadherin by adenoviral gene transfer in N-cadherin-expressing melanoma cells restored gap junctional compatibility with keratinocytes. Our data suggest that (1) melanocyte transformation is associated with loss of the pre-existing gap junctional activity with keratinocytes but a concomitant gain of communication with a newly juxtaposed cell type, the fibroblasts, (2) the specificity of gap junctional formation during melanoma development is determined by the cadherin profile on the melanocytic cells and (3) the overall gap junctional activity of melanocytic cells is not reduced with transformation.

The effects of a unilateral ultrasound-guided renal biopsy on renal function in healthy sedated cats.

Complications of renal biopsies are well documented except for the change in renal function after a biopsy. Eighteen healthy, adult cats were divided into two groups (n = 9 cats/group). For the measurement of global and split renal function, Group 1 used the renal uptake of 99mTc-DTPA and Group 2 used the renal uptake of 99mTc-MAG3. Scintigraphic data were collected on days (-4), (-3), 0, 1, 2, and 4 post renal biopsy. Using ultrasound guidance, biopsies were taken from the right renal cortex on dO, before acquiring scintigraphic images. P - values less than 0.10 were considered significant due to the limited number of observations. The only statistically significant change (p = 0.08) in global renal function detected was by day following a unilateral renal biopsy. Cats imaged using 99mTc-MAG3 had discernible liver activity. A unilateral, ultrasound guided renal biopsy has minimal effect on renal function in normal, healthy sedated cats.

Ras signaling through PI3K confers hormone-independent proliferation that is compatible with differentiation.

Hormones are specialized mitogens that stimulate proliferation in their differentiated target cells. Thyrotropin (TSH), the physiologic regulator of thyroid cells, stimulates cAMP-mediated proliferation and thyroid-specific gene expression. The mitogenic effects of TSH require Ras, therefore Ras activation should be compatible with the maintenance of thyroid differentiation. However, expression of activated Ras extinguishes the differentiated phenotype of thyroid cells. One explanation for this apparent paradox is the selective utilization of Ras effector pathways. We tested the hypothesis that Ras signaling through PI3K mediates the mitogenic effects of TSH in cells which retain their differentiated character. Expression of a Ras effector mutant (RasV12S35) that signals preferentially through Raf-1, although sufficient to confer TSH-independent proliferation, abolished hormone-regulated expression of thyroglobulin and the sodium/iodide symporter. In contrast, expression of a Ras mutant (RasV12C40) that binds selectively to PI3K conferred TSH-independent proliferation without marked effects on thyroid-specific gene expression. Unlike the inhibitory effects of TSH on the proliferation of RasV12S35-expressing cells, TSH enhanced RasV12C40-stimulated proliferation by further increasing the activity of p70s6k, an important mediator of the mitogenic effects of TSH and RasV12C40. These results demonstrate that channeling Ras-dependent signals to PI3K confers TSH with the ability to stimulate proliferation in differentiated cells. Oncogene (2000) 19, 924 - 932.

Differential effects of acute and chronic exposure to interferon-gamma on cyclic adenosine 3',5'-monophosphate response element-regulated gene expression.

TSH stimulates proliferation and maintains differentiated function in thyroid follicular cells. The mitogenic activity and the stimulatory effects of TSH on thyroid-specific gene expression are impaired by interferon-gamma (IFNgamma); however, the mechanisms for these effects have not been elucidated in detail. We examined the effects of IFNgamma on acute responses to TSH in rat thyroid cells. IFNgamma did not impair TSH-stimulated p70/p85 ribosomal protein S6 kinase (p70/p85s6k) activity or cAMP response element (CRE)-regulated gene expression, although it inhibited DNA synthesis and thyroglobulin expression, effects measured over a more prolonged time course than those on kinase activity and reporter gene expression. Unexpectedly, when cells were chronically exposed to IFNgamma, CRE-lacZ promoter activity was decreased, whereas other cAMP-mediated signals, such as p70/p85s6k activity and CRE-binding protein phosphorylation, were unaffected. Activating protein-1-regulated promoters were also impaired by IFNgamma treatment, but with kinetics that differed from those of CRE-regulated promoters. Neither acute nor chronic treatment with interleukin-1beta impaired cAMP signaling, indicating that the effects of IFNgamma are specific. These studies identify CRE- and activating protein-1-regulated promoters as targets of IFNgamma in thyroid cells and fibroblasts. IFNgamma-mediated inhibition of these promoters, in addition to those containing thyroid-specific transcription factor-1-binding sites, may contribute to the profound effects of IFNgamma on thyroid cells.

There are at least three genetically distinct small piroplasms from dogs.

The 18S nuclear subunit ribosomal RNA (18S rRNA) gene of small piroplasms isolated from dogs from Okinawa (Japan), Oklahoma, North Carolina, Indiana, Missouri, and Alabama, was isolated and sequenced. Phylogenetic analysis of these sequences and comparisons with sequences from other Babesia, Cytauxzoon, and Theileria species revealed that all canine small babesial isolates, with the exception of isolates from California and Spain, were placed in a group containing the Babesia spp. sensu stricto. Within the Babesia spp. sensu stricto, there was support for separating the small canine piroplasms from the large canine piroplasm, Babesia canis. The isolate from California was in a distinct phylogenetic clade, closely related to babesial isolates from wildlife and humans from the Western US. The canine isolate from Spain was closely related to Babesia microti. These results suggest that there are multiple small piroplasm species in dogs. The isolates from the Midwestern and Eastern US and the one from Japan probably represent a single species with wide geographic distribution.

Atypical protein kinase C-zeta stimulates thyrotropin-independent proliferation in rat thyroid cells.

Several reports have indicated that protein kinase C (PKC) is an important regulator of proliferation in thyroid cells. Unlike TSH, the mitogenic effects of phorbol esters are accompanied by de-differentiation. The role of individual PKC isoforms in thyroid cell proliferation and differentiation has not been examined. Recent studies have implicated the atypical PKCzeta, a phorbol ester-unresponsive isozyme, in cell proliferation, death, and survival. We overexpressed PKCzeta in Wistar rat thyroid (WRT) cells and determined that PKCzeta conferred TSH-independent DNA synthesis and cell proliferation. Cells overexpressing PKCzeta show higher levels of phosphorylated p42/p44 MAPK compared with vector-transfected cells. Experiments using a luciferase reporter for Elk-1 revealed that PKCzeta overexpressing cells exhibit higher basal Elk-1 transcriptional activity than vector-transfected control cells. Interestingly, stimulation of Elk-1 transcriptional activity by MEK1, a p42/p44 MAPK kinase, was significantly enhanced in cells overexpressing PKCzeta. Strikingly, TSH retained the ability to stimulate Tg expression in cells expressing PKCzeta. These results suggest that PKCzeta stimulates TSH-independent mitogenesis through a p42/p44 MAPK-dependent pathway. Unlike overexpression of Ras or phorbol ester treatment, PKC overexpression does not impair thyroglobulin (Tg) expression.

Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation.

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.

Prostaglandin F2alpha (PGF2alpha) and the isoprostane, 8, 12-iso-isoprostane F2alpha-III, induce cardiomyocyte hypertrophy. Differential activation of downstream signaling pathways.

Prostaglandin receptors may be activated by their cognate ligand or by free radical catalyzed isoprostanes, products of arachidonic acid peroxidation. For example, prostaglandin F2alpha (PGF2alpha) causes hypertrophy of neonatal rat ventricular myocytes, via the PGF2alpha receptor (FP). However, the FP may also be activated by the isoprostane, 8,12-iso-iPF2alpha-III (Kunapuli, P., Lawson, J. A., Rokach, J., and FitzGerald, G. A. (1997) J. Biol. Chem. 272, 27147-27154). Both ligands induce myocyte hypertrophy with overlapping potencies. Interestingly, the hypertrophic effects of these two agonists on cardiomyocytes are additive. Furthermore, the preference of these two agonists for activation of intracellular signal transduction pathways differs in several respects. Thus, PGF2alpha and 8,12-iso-iPF2alpha-III stimulate inositol phosphate formation with EC50 values of 50 +/- 12 nM and 3.5 +/- 0.6 microM, respectively. Moreover, PGF2alpha causes a robust activation ( approximately 50-fold) of Erk2, whereas 8,12-iso-iPF2alpha-III has no effect. Similarly, PGF2alpha causes translocation of cytosolic phospholipase A2 and also results in a 7-fold increment in the formation of 6-keto-PGF1alpha, whereas 8,12-iso-iPF2alpha-III exerts no effect on this pathway. On the other hand, both agonists are equally potent in activating JNK1 and c-Jun, whereas neither activates the p38 kinase. Both PGF2alpha and 8,12-iso-iPF2alpha-III activate the p70S6 kinase (p70(S6K)), but not Akt, downstream of phosphatidylinositol-3-kinase (PI3K). However, both wortmannin, a PI3K inhibitor, and rapamycin, an inhibitor of p70(S6K) activity, inhibit 8,12-iso-iPF2alpha-III -induced myocyte hypertrophy, with IC50 values of 60 +/- 12 and 3 +/- 1.7 nM, respectively, whereas neither compound abrogates the PGF2alpha-mediated response. Thus, both PGF2alpha and 8,12-iso-iPF2alpha-III induce myocyte hypertrophy via discrete signaling pathways. Although both agonists signal via the JNK pathway to initiate changes in c-Jun-dependent gene transcription, PGF2alpha preferentially activates the MEK-Erk2- cytosolic phospholipase A2 pathway. In contrast, the PI3K-p70(S6K) pathway appears to be essential for 8,12-iso-iPF2alpha-III-induced myocyte hypertrophy.

Differential effects of protein kinase A on Ras effector pathways.

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35, a mutant which binds Raf, or RasG37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. RasS35 stimulated morphological transformation and anchorage-independent growth. RasG37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exerted differential effects on TSH signaling. RasS35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.

Differential effects of cyclic adenosine 3',5'-monophosphate on p70 ribosomal S6 kinase.

cAMP exerts differential effects on mitogenic signaling pathways. In many cells, cAMP inhibits growth factor-stimulated MAPK activity and proliferation. In others, cAMP promotes growth. TSH stimulates proliferation through elevations in cAMP in thyroid follicular cells. This mitogenic pathway is dependent upon both protein kinase A and Ras, but not upon Raf-1, mitogen-activated protein kinase kinase, or mitogen-activated protein kinase. We report that TSH, acting through cAMP, activates pp70s6k and that this activity is required for TSH-stimulated DNA synthesis. A similar role for pp70s6k in cAMP-mediated mitogenesis was observed in secondary rat Schwann cells and in Swiss3T3 fibroblasts, two additional cell types that respond to cAMP with growth. In contrast, cAMP elevation did not activate pp70s6k in NIH3T3 or REF52 fibroblasts, cells in which cAMP fails to stimulate proliferation. Together, these results suggest that pp70s6k plays an important and general role in cAMP-mediated proliferation.