Nonclassical Target Tissues Research Articles

Vitamin D: Structure and mechanism of action

Vitamin D has received more attention in recent years as a result of the resurgence of vitamin D deficiency and rickets as a global health issue, as well as compelling evidence in the laboratory indicating that 1,25-dihydroxy vitamin D3 [1,25(OH)2D3], the hormonally active form of vitamin D, generates several extraskeletal biological responses such as inhibition of breast, colon, and prostate cancer cell progression; effects on the cardiovascular system; and protects against. This review covers our present knowledge of vitamin D and its bioactivation, as well as fresh findings that have altered our understanding of vitamin D activity in both classical and nonclassical target tissues. This page also assesses vitamin D's alleged involvement in extraskeletal health, provides an overview of 1,25(OH)2D3 analogs that have been created, and highlights unanswered problems.

Estrogen Receptor Beta in Cancer: an Attractive Target for Therapy

While it is well documented that the mitogenic actions of estrogens are critical in the development and progression of human breast and some gynecologic cancers, only latest data demonstrate a crucial involvement of estrogen-signaling in the carcinogenesis of non-classical estrogen target tissues, as colon, prostate, lung, skin, and brain. Only recently it has also been found out that the biological effects of estrogens are mediated by two distinct estrogen receptors (ERs), ERα and ERβ, and that their relative levels in a given cell are important determinants of response to estradiol and selective estrogen receptor modulators. Indeed, although ERα and ERβ have similar structure, they produce different effects, and there is currently increasing evidence that, for some tumors, an imbalanced ERβ expression might play a pivotal role in tumor development and progression. However, the prognostic value, the potential significance in predicting response to endocrine therapy, and, eventually, the utility of ERβ as a therapeutic target need to be assessed in large-scale and prospective clinical studies. This review examines the experimental and clinical evidences for a role of ERβ in carcinogenesis of classical and nonclassical estrogen target tissues. If anomalies of ERβ expression could be demonstrated to represent a critical step in the development and progression of some types of cancers, its re-expression by genetic engineering, as well as the use of targeted ERβ therapies would constitute new important therapeutic approaches.

Extrarenal effects of aldosterone

The renal distal tubule has been considered for a long time as the main cellular target of aldosterone, where the hormone enhances sodium reabsorption and potassium secretion. However, other cell types in nonepithelial tissues, such as the heart, the vessels, adipose tissue, and macrophages, are now also recognized as targets for aldosterone. The functions that aldosterone exerts in these nonclassical target tissues are still a matter of debate. This review will highlight the recent findings on the extrarenal effects of aldosterone. Numerous studies showed that aldosterone exerts profibrotic and proinflammatory effects, but one or more cofactors such as salt, angiotensin II, and oxidative stress are required. Moreover, inflammation and macrophage infiltration are a prerequisite to aldosterone-induced cardiac fibrosis. This underlines a key role for aldosterone and the mineralocorticoid receptor in macrophages. Inflammatory effects of aldosterone in vascular smooth muscle cells involve trafficking to lipid rafts/caveolae through receptor tyrosine kinases. Finally, a growing body of evidence indicates a prominent role of aldosterone/mineralocorticoid receptor in the metabolic syndrome, in insulin resistance, and in adipocyte biology. The idiom from Socrates, 'the more we learn, the less we know', can be applied to aldosterone with its different facets and its pleiotropic effects. There is clear evidence for rapid nongenomic effects of aldosterone, mineralocorticoid receptor-dependent and mineralocorticoid receptor-independent signaling, in the heart, the vessels, and other nonepithelial tissues, leading to inflammation, fibrosis, and progression of cardiovascular diseases including hypertension and metabolic syndrome.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Rescue of Obesity-Induced Infertility in Female Mice due to a Pituitary-Specific Knockout of the Insulin Receptor

Obesity is associated with insulin resistance in metabolic tissues such as adipose, liver, and muscle, but it is unclear whether nonclassical target tissues, such as those of the reproductive axis, are also insulin resistant. To determine if the reproductive axis maintains insulin sensitivity in obesity in vivo, murine models of diet-induced obesity (DIO) with and without intact insulin signaling in pituitary gonadotrophs were created. Diet-induced obese wild-type female mice (WT DIO) were infertile and experienced a robust increase in luteinizing hormone (LH) after gonadotropin-releasing hormone (GnRH) or insulin stimulation. By contrast, both lean and obese mice with a pituitary-specific knockout of the insulin receptor (PitIRKO) exhibited reproductive competency, indicating that insulin signaling in the pituitary is required for the reproductive impairment seen in DIO and that the gonadotroph maintains insulin sensitivity in a setting of peripheral insulin resistance.

Vitamin D Deficiency Promotes Human Breast Cancer Growth in a Murine Model of Bone Metastasis

Vitamin D exerts antiproliferative, prodifferentiation, and proapoptotic effects on nonclassic target tissues such as breast. Blood levels of 25-hydroxyvitamin D [25(OH)D], the most sensitive indicator of vitamin D status, are inversely correlated with breast cancer risk; however, a causal relationship between vitamin D deficiency and breast cancer growth in bone has not been assessed. We examined the effect of vitamin D deficiency on the intraskeletal growth of the human breast cancer cell line MDA-MB-231-TxSA in a murine model of malignant bone lesions. Subsets of mice were treated concurrently with osteoprotegerin (OPG) to abrogate bone resorption. Outcomes were assessed by repeated radiographic and end-point micro-computed tomography and histologic analyses. Mice weaned onto a vitamin D-free diet developed vitamin D deficiency within 4 weeks [mean +/- SE serum 25(OH)D: 11.5 +/- 0.5 nmol/L], which was sustained throughout the study and was associated with secondary hyperparathyroidism and accelerated bone turnover. Osteolytic lesions appeared earlier and were significantly larger in vitamin D-deficient than in vitamin D-sufficient mice after 2 weeks (radiographic osteolysis: +121.5%; histologic tumor area: +314%; P < 0.05). Although OPG treatment reduced the size of radiographic osteolyses and tumor area in both groups, tumors remained larger in OPG-treated vitamin D-deficient compared with OPG-treated vitamin D-sufficient mice (0.53 +/- 0.05 mm(2) versus 0.19 +/- 0.05 mm2; P < 0.05). We conclude that vitamin D deficiency promotes the growth of human breast cancer cells in the bones of nude mice. These effects are partly mediated through secondary changes in the bone microenvironment, along with direct effects of vitamin D on tumor growth.

Role of Mitochondrial Dysfunction in Insulin Resistance

Insulin resistance is characteristic of obesity, type 2 diabetes, and components of the cardiometabolic syndrome, including hypertension and dyslipidemia, that collectively contribute to a substantial risk for cardiovascular disease. Metabolic actions of insulin in classic insulin target tissues (eg, skeletal muscle, fat, and liver), as well as actions in nonclassic targets (eg, cardiovascular tissue), help to explain why insulin resistance and metabolic dysregulation are central in the pathogenesis of the cardiometabolic syndrome and cardiovascular disease. Glucose and lipid metabolism are largely dependent on mitochondria to generate energy in cells. Thereby, when nutrient oxidation is inefficient, the ratio of ATP production/oxygen consumption is low, leading to an increased production of superoxide anions. Reactive oxygen species formation may have maladaptive consequences that increase the rate of mutagenesis and stimulate proinflammatory processes. In addition to reactive oxygen species formation, genetic factors, aging, and reduced mitochondrial biogenesis all contribute to mitochondrial dysfunction. These factors also contribute to insulin resistance in classic and nonclassic insulin target tissues. Insulin resistance emanating from mitochondrial dysfunction may contribute to metabolic and cardiovascular abnormalities and subsequent increases in cardiovascular disease. Furthermore, interventions that improve mitochondrial function also improve insulin resistance. Collectively, these observations suggest that mitochondrial dysfunction may be a central cause of insulin resistance and associated complications. In this review, we discuss mechanisms of mitochondrial dysfunction related to the pathophysiology of insulin resistance in classic insulin-responsive tissue, as well as cardiovascular tissue.

A neuroendocrine-immune interface. The immunostimulatory state of aldosteronism.

Hypertension is an important determinant of heart failure. Ventricular systolic and/or diastolic dysfunction can eventuate in an activation of the renin-angiotensin-aldosterone system (RAAS). Circulating RAAS effector hormones lead to: the appearance of the congestive heart failure syndrome; and a systemic illness that features oxi/nitrosative stress in various tissues, including blood, together with circulating pro-inflammatory cytokines, anorexia and, ultimately, cachexia. In addition to its well-known endocrine properties, expressed in epithelial cells of classic target tissues, aldosterone (ALDO) has an emerging portfolio of actions in nonclassic target tissues, such as circulating lymphocytes and monocytes (or peripheral blood mononuclear cells [PBMC]). This neuroendocrine-immune interface is based on Na(+)-dependent, ALDO-induced iterations in PBMC cytosolic free [Mg(2+)](i) and [Ca(2+)](i). Ca(2+) loading contributes to an induction of oxi/nitrosative stress and activation of PBMC transcriptome. This immunostimulatory state begets a pro-inflammatory/fibrogenic vascular phenotype involving the heart and systemic organs and can be prevented by either ALDO receptor antagonism or antioxidant. The established efficacy of ALDO receptor antagonism as an integral component of the overall management of symptomatic heart failure may include its immunomodulatory properties. This brief review traces studies that led to and then identified the neuroendocrine-immune interface that accompanies aldosteronism.

The distribution of estrogen receptor beta is distinct to that of estrogen receptor alpha and the androgen receptor in human skin and the pilosebaceous unit.

Both estrogens and androgens play important parts in skin and hair physiology, although studies of estrogen action in human skin have been rather limited. Recently, a second estrogen receptor (beta) has been identified in many nonclassical target tissues, including androgen-dependent tissues. Therefore, we have revisited the role of estrogens in human skin and hair by comparing the pattern of expression by immunohistochemistry for both estrogen receptors (alpha and beta) and the androgen receptor. Immunolocalization of androgen receptors was only seen in hair follicle dermal papilla cells and the basal cells of the sebaceous gland. Little specific staining of estrogen receptor alpha was seen anywhere except the sebaceous gland. In contrast estrogen receptor beta was highly expressed in epidermis, blood vessels, and dermal fibroblasts, whereas in the hair follicle it was localized to nuclei of the outer root sheath, epithelial matrix, and dermal papilla cells. Serial sections also showed strong nuclear expression of estrogen receptor beta in the cells of the bulge, whereas neither estrogen receptor alpha or androgen receptor was expressed. In the sebaceous gland, estrogen receptor beta was expressed in both basal and partially differentiated sebocytes in a similar pattern to estrogen receptor alpha. There was no obvious difference in the expression of either estrogen receptor in male or female nonbalding scalp skin. The results of this immunohistochemical study propose that estrogen receptor beta and not estrogen receptor alpha is the main mediator of estrogen action in human skin and the hair follicle. Further studies with androgen-dependent skin are required to determine whether estrogen receptor beta has a regulatory role on androgen receptor expression in the hair follicle in parallel with its role in other androgen-dependent tissues.

Tissue-specific targeting of the insulin receptor gene.

The techniques to study the mechanisms that underlie the pathogenesis of disease processes have been revolutionized by the development of methods that allow spatiotemporal control of gene deletion or gene expression in transgenic and knockout animals. The ability to interfere with the function of a single protein in a specific tissue allows unprecedented flexibility for exploring gene function in both health and disease. The present review will summarize some of the different knockouts and transgenics generated recently to study type 2 diabetes and critically evaluate the techniques used to examine the function of the insulin receptor in two nonclassical insulin target tissues--the pancreatic islet and the central nervous system.

Development of an ER Action Indicator Mouse for the Study of Estrogens, Selective ER Modulators (SERMs), and Xenobiotics

We have developed a transgenic mouse that functions as a reporter of ER activity, termed ER action indicator (ERIN), by incorporating a transgene with an estrogen-responsive promoter (three copies of the vitellogenin estrogen response element with a minimal thymidine kinase promoter) linked to the reporter gene β-galactosidase. Evaluation of ER activity in female ERIN mice demonstrated estrogen-inducible expression of the reporter gene in the uterus, pituitary, and hypothalamus; established targets of estrogen action. Importantly, we also identified ER activity in a number of nonclassical estrogen target tissues, including kidney, liver, adrenal, and thyroid gland. ERIN provides a system to measure the same end point (transgene regulation) in different target tissues, permitting separation of the contributions of cell- and promoter-specific factors in determining ER pharmacology. In this regard we observed that on this specific promoter the pituitary gland was 25-fold more sensitive than the uterus to the estrogen diethylstilbestrol, implying the existence of cell-specific factors that influence ligand sensitivity. Our studies also identified considerable difference in the efficacy and potency of ER ligands in the uterus when ER transcriptional activity was assayed vs. uterine weight gain. Specifically, we observed that the environmental estrogen bisphenol A was a potent agonist in stimulating ER transcriptional activity, whereas it exhibited little uterotropic activity. In contrast to bisphenol A, tamoxifen significantly increased uterine weight, but minimally induced ER reporter activity in this tissue. Given the results of these studies, we believe that ERIN will be a useful model to evaluate ER ligand pharmacology and will assist in defining the cellular and molecular mechanisms that determine agonist and antagonist activity.

Development of an ER action indicator mouse for the study of estrogens, selective ER modulators (SERMs), and Xenobiotics.

We have developed a transgenic mouse that functions as a reporter of ER activity, termed ER action indicator (ERIN), by incorporating a transgene with an estrogen-responsive promoter (three copies of the vitellogenin estrogen response element with a minimal thymidine kinase promoter) linked to the reporter gene beta-galactosidase. Evaluation of ER activity in female ERIN mice demonstrated estrogen-inducible expression of the reporter gene in the uterus, pituitary, and hypothalamus; established targets of estrogen action. Importantly, we also identified ER activity in a number of nonclassical estrogen target tissues, including kidney, liver, adrenal, and thyroid gland. ERIN provides a system to measure the same end point (transgene regulation) in different target tissues, permitting separation of the contributions of cell- and promoter-specific factors in determining ER pharmacology. In this regard we observed that on this specific promoter the pituitary gland was 25-fold more sensitive than the uterus to the estrogen diethylstilbestrol, implying the existence of cell-specific factors that influence ligand sensitivity. Our studies also identified considerable difference in the efficacy and potency of ER ligands in the uterus when ER transcriptional activity was assayed vs. uterine weight gain. Specifically, we observed that the environmental estrogen bisphenol A was a potent agonist in stimulating ER transcriptional activity, whereas it exhibited little uterotropic activity. In contrast to bisphenol A, tamoxifen significantly increased uterine weight, but minimally induced ER reporter activity in this tissue. Given the results of these studies, we believe that ERIN will be a useful model to evaluate ER ligand pharmacology and will assist in defining the cellular and molecular mechanisms that determine agonist and antagonist activity.

Regulation of Metallothionein II Messenger Ribonucleic Acid Measures Exogenous Estrogen Receptor-β Activity in SAOS-2 and LNCaPLN3 Cells

Estrogen receptor-beta (ERbeta) is a recently discovered member of the steroid hormone superfamily. Because its distribution is distinct from that of the classical estrogen receptor, and it is expressed in several nonclassical estrogen target tissues (e.g. prostate and bladder), its role in mediating the action of estrogen is unclear. One approach to elucidating the function of this receptor is to identify genes that it regulates. Using differential display, we profiled the messenger RNAs regulated by 17beta-estradiol in SAOS-2 and LNCaPLN3 cells overexpressing ERbeta. Follow-up studies used cells expressing either ERalpha or ERbeta. One gene, metallothionein II, was regulated by both receptor subtypes in LNCaPLN3 cells, but only by ERbeta in SAOS-2 cells. Because cycloheximide blocks this response, induction is probably mediated through regulation of at least one other protein. Identification of endogenous genes that are regulated differentially by ERalpha and ERbeta will be valuable tools in elucidating the function of ERbeta and the mechanisms by which these two receptors regulate transcription.

Introduction

Introduction

Vitamin D: A Hormonal Regulator of the cAMP Signaling Pathway

In the rat thyroid FRTL-5 cell line calcitriol, the biologically most active of the naturally occurring vitamin D metabolites, attenuates both TSH-stimulated cAMP production and the effects of cAMP. Calcitriol treatment abolishes the upregulation of the TSHR number occurring in cells cultivated in the absence of TSH. In addition, the level of G(i-2)alpha increases, which may further attenuate the transmembrane signaling of TSH and facilitate the effects of IGFs. The effect of cAMP on PKAI stimulation is inhibited by increasing the level of the PKA subunit RIIbeta. Regulation of TSHR, G(i-2)alpha and RIIbeta is associated with altered cell proliferation and differentiation in several cells and tissues. Effects of calcitriol on these proteins indicate how the vitamin D endocrine system may regulate cAMP signaling in both classical and nonclassical target tissues.

Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines.

PTH and PTH-related peptides (PTHrPs) interact with a common PTH/PTHrP receptor (type I), which is expressed in many tissues, including bone and kidney. Amino-terminal PTH and PTHrPs also recognize receptors in several nonclassical PTH target tissues, and in some of these, the signaling mechanisms differ qualitatively from those of the classical type I receptor. In normal keratinocytes and squamous carcinoma cell lines, PTH and PTHrP stimulate a rise in intracellular calcium, but not cAMP, suggesting the existence of an alternate, type II PTH/PTHrP receptor. SqCC/Y1 squamous carcinoma cells stably expressing the type I receptor displayed sensitive intracellular cAMP responses to PTHrP and PTH, indicating that these cells express functional GS proteins and that the type I receptor is capable of signaling through adenylyl cyclase in this cell line. Therefore, the endogenous type II receptor in SqCC/Y1 cells differs from the cloned type I receptor. We next examined whether messenger RNA (mRNA) from keratinocytes and squamous cell lines could hybridize to a human type I PTH/PTHrP receptor complementary DNA [1.9 kilobases (kb)]. No type I receptor mRNA (2.3 kb) was detected in polyadenylated RNA from any of the squamous cell lines. However, squamous cell lines did express several mRNA transcripts that hybridized with the type I receptor probe, yet were smaller (1 and 1.5 kb) or larger (3.5-5 kb) than the cloned receptor mRNA. The predominant mRNA in two squamous carcinoma cell lines and normal keratinocytes was a 1-kb transcript. Northern analysis with five different region-specific probes that span the entire coding region of the human type I receptor was used to map homologous regions within each of the transcripts. Several of the transcripts identified in squamous lines are also present in polyadenylated RNA from SaOS-2 human bone cells, but a unique 1-kb transcript hybridizing to probe 2 (nucleotides 490-870) was observed only in squamous cells. The smaller 1- and 1.5-kb transcripts did not hybridize to probes corresponding to the extreme 5'- and 3'-coding regions of the type I receptor complementary DNA. Ribonuclease protection analysis employing riboprobes that correspond to the five region-specific DNA probes revealed strong RNA signals of the expected size in SaOS-2 cells, but no hybridization with squamous cell RNA. Several smaller, but minor, bands that were unique to squamous cells were observed with riboprobe 2 only, suggesting partial homology of this region with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines

PTH and PTH-related peptides (PTHrPs) interact with a common PTH/PTHrP receptor (type I), which is expressed in many tissues, including bone and kidney. Amino-terminal PTH and PTHrPs also recognize receptors in several nonclassical PTH target tissues, and in some of these, the signaling mechanisms differ qualitatively from those of the classical type I receptor. In normal keratinocytes and squamous carcinoma cell lines, PTH and PTHrP stimulate a rise in intracellular calcium, but not cAMP, suggesting the existence of an alternate, type II PTH/PTHrP receptor. SqCC/Y1 squamous carcinoma cells stably expressing the type I receptor displayed sensitive intracellular cAMP responses to PTHrP and PTH, indicating that these cells express functional GS proteins and that the type I receptor is capable of signaling through adenylyl cyclase in this cell line. Therefore, the endogenous type II receptor in SqCC/Y1 cells differs from the cloned type I receptor. We next examined whether messenger RNA (mRNA) from keratinocytes and squamous cell lines could hybridize to a human type I PTH/PTHrP receptor complementary DNA [1.9 kilobases (kb)]. No type I receptor mRNA (2.3 kb) was detected in polyadenylated RNA from any of the squamous cell lines. However, squamous cell lines did express several mRNA transcripts that hybridized with the type I receptor probe, yet were smaller (1 and 1.5 kb) or larger (3.5-5 kb) than the cloned receptor mRNA. The predominant mRNA in two squamous carcinoma cell lines and normal keratinocytes was a 1-kb transcript. Northern analysis with five different region-specific probes that span the entire coding region of the human type I receptor was used to map homologous regions within each of the transcripts. Several of the transcripts identified in squamous lines are also present in polyadenylated RNA from SaOS-2 human bone cells, but a unique 1-kb transcript hybridizing to probe 2 (nucleotides 490-870) was observed only in squamous cells. The smaller 1- and 1.5-kb transcripts did not hybridize to probes corresponding to the extreme 5'- and 3'-coding regions of the type I receptor complementary DNA. Ribonuclease protection analysis employing riboprobes that correspond to the five region-specific DNA probes revealed strong RNA signals of the expected size in SaOS-2 cells, but no hybridization with squamous cell RNA. Several smaller, but minor, bands that were unique to squamous cells were observed with riboprobe 2 only, suggesting partial homology of this region with the type I receptor.

Alteration of phosphotyrosine phosphatase activity in tissues from diabetic and pregnant rats

Membrane-associated tyrosine phosphatase activities were studied in two distinct states of insulin resistance: diabetes and pregnancy. Using a novel immunoenzymatic assay with intact insulin-like growth factor-I (IGF-I) and insulin receptors as substrates, we show that phosphotyrosine-protein phosphatases (PTP-ases) from normal rat tissues induce a decrease in tyrosine phosphorylation of both receptors. Membrane fractions from kidney, brain, and liver contain the highest PTP-ase activity toward the insulin receptor. After 20-day streptozotocin-induced diabetes, PTP-ase activities are increased by 70% in the placenta, reduced by 40-50% in liver and skeletal muscle, and remained unchanged in the nonclassical insulin target tissues, kidney and brain. In general, the dephosphorylation of IGF-I receptor follows a pattern similar to that of insulin receptor except in red skeletal muscle in which it is not modified. Pregnancy also induces alterations of liver PTP-ases similar to those elicited by diabetes with a 50% reduction of insulin and IGF-I receptor dephosphorylation. This effect of pregnancy is further potentiated by diabetes. The alterations in the activity of hepatic PTP-ases from diabetic and pregnant rats are associated with a decreased autophosphorylation of the insulin receptor, suggesting that the diminution of phosphatase activity might be associated to the state of receptor phosphorylation and activation. Our data demonstrate that alterations of PTP-ases in insulin target tissues are found in two insulin-resistant states, one characterized by hyperinsulinemia, pregnancy and one by insulinopenia, streptozotocin-diabetes. These observations suggest a possible relationship between the defective activity of receptor tyrosine kinases and membrane-associated phosphatases from insulin responsive tissues.

Alteration of phosphotyrosine phosphatase activity in tissues from diabetic and pregnant rats.

Membrane-associated tyrosine phosphatase activities were studied in two distinct states of insulin resistance: diabetes and pregnancy. Using a novel immunoenzymatic assay with intact insulin-like growth factor-I (IGF-I) and insulin receptors as substrates, we show that phosphotyrosine-protein phosphatases (PTP-ases) from normal rat tissues induce a decrease in tyrosine phosphorylation of both receptors. Membrane fractions from kidney, brain, and liver contain the highest PTP-ase activity toward the insulin receptor. After 20-day streptozotocin-induced diabetes, PTP-ase activities are increased by 70% in the placenta, reduced by 40-50% in liver and skeletal muscle, and remained unchanged in the nonclassical insulin target tissues, kidney and brain. In general, the dephosphorylation of IGF-I receptor follows a pattern similar to that of insulin receptor except in red skeletal muscle in which it is not modified. Pregnancy also induces alterations of liver PTP-ases similar to those elicited by diabetes with a 50% reduction of insulin and IGF-I receptor dephosphorylation. This effect of pregnancy is further potentiated by diabetes. The alterations in the activity of hepatic PTP-ases from diabetic and pregnant rats are associated with a decreased autophosphorylation of the insulin receptor, suggesting that the diminution of phosphatase activity might be associated to the state of receptor phosphorylation and activation. Our data demonstrate that alterations of PTP-ases in insulin target tissues are found in two insulin-resistant states, one characterized by hyperinsulinemia, pregnancy and one by insulinopenia, streptozotocin-diabetes. These observations suggest a possible relationship between the defective activity of receptor tyrosine kinases and membrane-associated phosphatases from insulin responsive tissues.

Absence of calbindin-D28 expression in nonclassical 1,25-dihydroxyvitamin D targets: analysis by polymerase chain reaction.

CaBP-D28 mRNA expression in rat heart, testis, and lung was assessed by polymerase chain reaction (PCR). The animal model used was the hyperinduced vitamin D-treated rat (100 ng 1,25-dihydroxyvitamin D subcutaneously, daily for 7 days). For the PCR studies, two pairs of 20 mer oligonucleotide primers (designated 1-4 according to their position on the coding strand, but with primers 3 and 4 in reverse orientation) derived from the rat CaBP-D28 cDNA sequence were tested in various combinations. Optimal conditions were established using a 1:100 dilution of cDNA from normal rat kidney. Bands of the predicted sizes of 869 (1, 3), 994 (1, 4), 725 (2, 3), and 850 (2, 4) nucleotide base pairs resulted, but with varying intensities: 2,4 approximately 1,3 > 1,4 > 2,3. Repeat PCR (recycling after 1:100 dilution and readdition of reagents and primers with at least one different primer) provided strong additional amplification, particularly with the 1,4/2,4 combination. Under these conditions, mixing experiments showed that CaBP-D28 transcripts were detectable at 10(-7)- to 10(-9)-fold lower levels of expression than in D+ kidney. When RNA was isolated and cDNA generated from test tissues from 4 individual vitamin D-stimulated (D+) and vitamin D-deficient (D-) rats, repeat PCR (1,4/2,4 primer combination) provided no evidence of significant CaBP-D28 mRNA expression in the nonclassic target tissues, in contrast to strong bands in both the D- kidney (undiluted) and D+ kidney (1:100 dilution) preparations.(ABSTRACT TRUNCATED AT 250 WORDS)