Nancy E. Cooke

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein

A line of mice deficient in vitamin D binding protein (DBP) was generated by targeted mutagenesis to establish a model for analysis of DBPs biological functions in vitamin D metabolism and action. On vitamin D-replete diets, DBP-/- mice had low levels of total serum vitamin D metabolites but were otherwise normal. When maintained on vitamin D-deficient diets for a brief period, the DBP-/-, but not DBP+/+, mice developed secondary hyperparathyroidism and the accompanying bone changes associated with vitamin D deficiency. DBP markedly prolonged the serum half-life of 25(OH)D and less dramatically prolonged the half-life of vitamin D by slowing its hepatic uptake and increasing the efficiency of its conversion to 25(OH)D in the liver. After an overload of vitamin D, DBP-/- mice were unexpectedly less susceptible to hypercalcemia and its toxic effects. Peak steady-state mRNA levels of the vitamin D-dependent calbindin-D9K gene were induced by 1,25(OH)2D more rapidly in the DBP-/- mice. Thus, the role of DBP is to maintain stable serum stores of vitamin D metabolites and modulate the rates of its bioavailability, activation, and end-organ responsiveness. These properties may have evolved to stabilize and maintain serum levels of vitamin D in environments with variable vitamin D availability.

The multifunctional properties and characteristics of vitamin D-binding protein

In recent years, our understanding of the many physiological, biochemical, and molecular functions and attributes of vitamin D-binding protein (DBP) has seen exciting and significant advances. Since its identification in 1959, many important functions of this abundant serum protein have been discovered. These range from the transport of vitamin D metabolites to possible roles in the immune system and host defense. With these discoveries, many questions regarding the biology of DBP have been raised and many remain to be answered. Our current understanding of the classic and less-recognized activities of DBP is discussed here.

A Defined Locus Control Region Determinant Links Chromatin Domain Acetylation with Long-Range Gene Activation

Gene activation in higher eukaryotes is often under the control of regulatory elements quite distant from their target promoters. It is unclear how such long-range control is mediated. Here we show that a single determinant of the human growth hormone locus control region (hGH LCR) located 14.5 kb 5prime prime or minute to the hGH-N promoter has a critical, specific, and nonredundant role in facilitating promoter trans factor binding and activating hGH-N transcription. Significantly, this same determinant plays an essential role in establishing a 32 kb acetylated domain that encompasses the entire hGH LCR and the contiguous hGH-N promoter. These data support a model for long-range gene activation via LCR-mediated targeting and extensive spreading of core histone acetylation.

Vitamin D-binding protein influences total circulating levels of 1,25-dihydroxyvitamin D3 but does not directly modulate the bioactive levels of the hormone in vivo.

Mice deficient in the expression of vitamin D-binding protein (DBP) are normocalcemic despite undetectable levels of circulating 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. We used this in vivo mouse model together with cells in culture to explore the impact of DBP on the biological activity of 1,25(OH)(2)D(3). Modest changes in the basal expression of genes involved in 1,25(OH)(2)D(3) metabolism and calcium homeostasis were observed in vivo; however, these changes seemed unlikely to explain the normal calcium balance seen in DBP-null mice. Further investigation revealed that despite the reduced blood levels of 1,25(OH)(2)D(3) in these mice, tissue concentrations were equivalent to those measured in wild-type counterparts. Thus, the presence of DBP has limited impact on the extracellular pool of 1,25(OH)(2)D(3) that is biologically active and that accumulates within target tissues. In cell culture, in contrast, the biological activity of 1,25(OH)(2)D(3) is significantly impacted by DBP. Here, although DBP deficiency had no effect on the activation profile itself, the absence of DBP strongly reduced the concentration of exogenous 1,25(OH)(2)D(3) necessary for transactivation. Surprisingly, analogous studies in wild-type and DBP-null mice, wherein we explored the activity of exogenous 1,25(OH)(2)D(3), produced strikingly different results as compared with those in vitro. Here, the carrier protein had virtually no impact on the distribution, uptake, activation profile, or biological potency of the hormone. Collectively, these experiments suggest that whereas DBP is important to total circulating 1,25(OH)(2)D(3) and sequesters extracellular levels of this hormone both in vivo and in vitro, the binding protein does not influence the hormones biologically active pool.

Characterization and histologic localization of human growth hormone-variant gene expression in the placenta.

The human growth hormone-variant (hGH-V) gene is one of five highly similar growth hormone-related genes clustered on the short arm of chromosome 17. Although the pattern of expression of the adjacent normal growth hormone (hGH-N) and chorionic somatomammotropin (hCS) genes in this cluster are well characterized, the expression of the hGH-V gene remains to be defined. In previous studies, we have demonstrated that the hGH-V gene is transcribed in the term placenta and expressed as two alternatively spliced mRNAs: one is predicted to encode a 22-kD hormone (hGH-V), the other retains intron 4 in its sequence resulting in the predicted synthesis of a novel 26-kD hGH-V-related protein (hGH-V2). In the present report, we document the expression of both of these hGH-V mRNA species in the villi of the term placenta, demonstrate an increase in their concentrations during gestation, and directly sublocalize hGH-V gene expression to the syncytiotrophoblastic epithelium of the term placenta by in situ cDNA-mRNA histohybridization. The demonstrated similarity in the developmental and tissue-specific expression of the hGH-V gene with that of the related hCS gene suggests that these two genes may share common regulatory elements.

Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns.

Stably transfected cell lines containing the normal human growth hormone (hGH-N) and human growth hormone-variant (hGH-V) genes have been established in order to study the expression of these two highly homologous genes. Each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse C127 cells. The transfected cells containing the hGH-N gene secrete two hGH proteins, 91% migrating at 22 kD and 9% migrating at 20 kD, the same relative proportions synthesized in vivo by the human pituitary. S1 nuclease analysis of mRNA from these cells confirms that 20 kD hGH is encoded by an alternatively spliced product of the primary hGH-N gene transcript in which the normal exon 3 splice-acceptor site is bypassed for a secondary site 15 codons within exon 3. Although the hGH-V gene is identical to the hGH-N gene for at least 15 nucleotides on either side of the normal and alternative exon 3 AG splice-acceptor sites, hGH-V synthesizes only a 22-kD protein. Reciprocal exchange of exon 3 and its flanking intron sequences between the hGH-N gene and the hGH-V gene, eliminates the synthesis of the 20-kD protein in both resultant chimeric genes. These results directly demonstrate that both the major 22-kD and the minor 20-kD forms of pituitary hGH are encoded by the alternative splicing products of a single hGH-N gene transcript. This alternative splicing is neither species nor tissue-specific and appears to be regulated by at least two separate regions remote from the AG splice-acceptor site.

Characterization of a human cDNA encoding a widely expressed and highly conserved cysteine-rich protein with an unusual zinc-finger motif

A human term placental cDNA library was screened at low stringency with a human prolactin cDNA probe. One of the cDNAs isolated hybridizes to a 1.8 kb mRNA present in all four tissues of the placenta as well as to every nucleated tissue and cell line tested. The sequence of the full-length cDNA was determined. An extended open reading frame predicted an encoded protein product of 20.5 kDa. This was directly confirmed by the in vitro translation of a synthetic mRNA transcript. Based upon the characteristic placement of cysteine (C) and histidine (H) residues in the predicted protein structure, this molecule contains four putative zinc fingers. The first and third fingers are of the C4 class while the second and fourth are of the C2HC class. Based upon sequence similarities between the first two and last two zinc fingers and sequence similarities to a related rodent protein, cysteine-rich intestinal protein (CRIP), these four finger domains appear to have evolved by duplication of a preexisting two finger unit. Southern blot analyses indicate that this human cysteine-rich protein (hCRP) gene has been highly conserved over the span of evolution from yeast to man. The characteristics of this protein suggest that it serves a fundamental role in cellular function.

Neurobeachin Is Essential for Neuromuscular Synaptic Transmission

We report a random disruption in the mouse genome that resulted in lethal paralysis in homozygous newborns. The disruption blocked expression of neurobeachin, a protein containing a BEACH (beige and Chediak-Higashi) domain implicated in synaptic vesicle trafficking and an AKAP (A-kinase anchor protein) domain linked to localization of cAMP-dependent protein kinase activity. nbea-null mice demonstrated a complete block of evoked synaptic transmission at neuromuscular junctions, whereas nerve conduction, synaptic structure, and spontaneous synaptic vesicle release were completely normal. These findings support an essential role for neurobeachin in evoked neurotransmitter release at neuromuscular junctions and suggest that it plays an important role in synaptic transmission.

Patterns of histone acetylation suggest dual pathways for gene activation by a bifunctional locus control region

The five genes of the human growth hormone (hGH) cluster are expressed in either the pituitary or placenta. Activation of the cluster is dependent on a locus control region (LCR) comprising pituitary‐ specific (HSI,II, −15 kb), placenta‐specific (HSIV, −30 kb) and shared (HSIII, −28 kb; HSV, −32 kb) DNase I hypersensitive sites. Gene activation in the pituitary is paralleled by acetylation of a 32 kb chromatin domain 5′ to the cluster centered at HSI,II. In the present study we observed that acetylation of this region in placental chromatin was discretely limited to shared HSIII and HSV. Transgenic studies revealed placenta‐specific activation of linked genes by a determinant (P‐element) located 2 kb 5′ to each of the four placentally expressed genes. A localized peak of histone acetylation was observed at these P‐elements in placenta but not pituitary. These data support a model for bifunctional action of the hGH LCR in which separate positive determinants, HSI,II and the P‐elements, activate their respective target genes by tissue‐specific recruitment of distinctly regulated histone acetyl transferase activities.

Bystander gene activation by a locus control region

Random assortment of genes within mammalian genomes establishes the potential for interference between neighboring genes with distinct transcriptional specificities. Long‐range transcriptional controls further increase this potential. Exploring this problem is of fundamental importance to understanding gene regulation. In the human genome, the Igβ (CD79b) gene is situated between the pituitary‐specific human growth hormone (hGH) gene and its locus control region (hGH LCR). Igβ protein is considered B‐cell specific; its only known role is in B‐cell receptor signaling. Unexpectedly, we found that hIgβ is transcribed at high levels in the pituitary. This Igβ transcription is dependent on pituitary‐specific epigenetic modifications generated by the hGH LCR. In contrast, expression of Igβ at its native site in B cells is independent of hGH LCR activity. These studies demonstrated that a gene with tissue‐restricted transcriptional determinants (B cell) can be robustly activated in an unrelated tissue (pituitary) due to fortuitous positioning within an active chromatin domain. This ‘bystander’ gene activation pathway impacts on current concepts of tissue specificity and models of active chromatin domains.