WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma.

High-level amplification of the protein phosphatase PPM1D (WIP1) is present in a subset of medulloblastomas (MBs) that have an expression profile consistent with active Sonic Hedgehog (SHH) signaling. We found that WIP1 overexpression increased expression of Shh target genes and cell proliferation in response to Shh stimulation in NIH3T3 and cerebellar granule neuron precursor cells in a p53-independent manner. Thus, we developed a mouse in which WIP1 is expressed in the developing brain under control of the Neurod2 promoter (ND2:WIP1). The external granule layer (EGL) in early postnatal ND2:WIP1 mice exhibited increased proliferation and expression of Shh downstream targets. MB incidence increased and survival decreased when ND2:WIP1 mice were crossed with an Shh-activated MB mouse model. Conversely, Wip1 knockout significantly suppressed MB formation in two independent mouse models of Shh-activated MB. Furthermore, Wip1 knockdown or treatment with a WIP1 inhibitor suppressed the effects of Shh stimulation and potentiated the growth inhibitory effects of SHH pathway-inhibiting drugs in Shh-activated MB cells in vitro. This suggests an important cross-talk between SHH and WIP1 pathways that accelerates tumorigenesis and supports WIP1 inhibition as a potential treatment strategy for MB.

YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog-dependent cerebellar granule neuron progenitor cells and medulloblastoma cells.

Postnatal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells of origin for the SHH-associated subgroup of medulloblastoma, is driven by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is upregulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh subgroup of medulloblastoma in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and MBCs and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

Oncogenic YAP promotes radioresistance and genomic instability in medulloblastoma through IGF2-mediated Akt activation.

Radiation therapy remains the standard of care for many cancers, including the malignant pediatric brain tumor medulloblastoma. Radiation leads to long-term side effects, whereas radioresistance contributes to tumor recurrence. Radio-resistant medulloblastoma cells occupy the perivascular niche. They express Yes-associated protein (YAP), a Sonic hedgehog (Shh) target markedly elevated in Shh-driven medulloblastomas. Here we report that YAP accelerates tumor growth and confers radioresistance, promoting ongoing proliferation after radiation. YAP activity enables cells to enter mitosis with un-repaired DNA through driving insulin-like growth factor 2 (IGF2) expression and Akt activation, resulting in ATM/Chk2 inactivation and abrogation of cell cycle checkpoints. Our results establish a central role for YAP in counteracting radiation-based therapies and driving genomic instability, and indicate the YAP/IGF2/Akt axis as a therapeutic target in medulloblastoma.

Divergent functions for eIF4E and S6 kinase by sonic hedgehog mitogenic signaling in the developing cerebellum.

Cerebellar development entails rapid peri-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for certain medulloblastomas. CGNPs require insulin-like growth factor (IGF) for survival and sonic hedgehog (Shh)-implicated in medulloblastoma-for proliferation. The IGF-responsive kinase mammalian target of rapamycin (mTOR) drives proliferation-associated protein synthesis. We asked whether Shh signaling regulates mTOR targets to promote CGNP proliferation despite constitutive IGF signaling under proliferative and differentiation-promoting conditions. Surprisingly, Shh promoted eukaryotic initiation factor 4E (eIF4E) expression, but inhibited S6 kinase (S6K). In vivo, S6K activity specifically marked the CGNP population transitioning from proliferation-competent to post-mitotic. Indeed, eIF4E was required for CGNP proliferation, while S6K activation drove cell cycle exit. Protein phosphatase 2A (PP2A) inhibition rescued S6K activity. Moreover, Shh upregulated the PP2A B56γ subunit, which targets S6K for inactivation and was required for CGNP proliferation. These findings reveal unique developmental functions for eIF4E and S6 kinase wherein their activity is specifically uncoupled by mitogenic Shh signaling.

Mitogenic Sonic hedgehog signaling drives E2F1-dependent lipogenesis in progenitor cells and medulloblastoma.

Deregulation of the Rb/E2F tumor suppressor complex and aberrantion of Sonic hedgehog (Shh) signaling are documented across the spectrum of human malignancies. Exaggerated de novo lipid synthesis is also found in certain highly proliferative, aggressive tumors. Here, we show that in Shh-driven medulloblastomas, Rb is inactivated and E2F1 is upregulated, promoting lipogenesis. Extensive lipid accumulation and elevated levels of the lipogenic enzyme fatty acid synthase (FASN) mark those tumors. In primary cerebellar granule neuron precursors (CGNPs), proposed Shh-associated medulloblastoma cells-of-origin, Shh signaling triggers E2F1 and FASN expression, whereas suppressing fatty acid oxidation (FAO), in a smoothened-dependent manner. In the developing cerebellum, E2F1 and FASN co-localize in proliferating CGNPs. in vivo and in vitro, E2F1 is required for FASN expression and CGNP proliferation, and E2F1 knockdown impairs Shh-mediated FAO inhibition. Pharmacological blockade of Rb inactivation and/or lipogenesis inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals In vivo. These findings identify a novel mechanism through which Shh signaling links cell cycle progression to lipid synthesis, through E2F1-dependent regulation of lipogenic enzymes. These findings pertinent to the etiology of tumor metabolism also underscore the key role of the Shh→E2F1→FASN axis in regulating de novo lipid synthesis in cancers, and as such its value as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors.

Sonic hedgehog (Shh) signal transduction via the G-protein-coupled receptor, Smoothened, is required for proliferation of cerebellar granule neuron precursors (CGNPs) during development. Activating mutations in the Hedgehog pathway are also implicated in basal cell carcinoma and medulloblastoma, a tumor of the cerebellum in humans. However, Shh signaling interactions with cell cycle regulatory components in neural precursors are poorly understood, in part because appropriate immortalized cell lines are not available. We have utilized primary cultures from neonatal mouse cerebella in order to determine (i) whether Shh initiates or maintains cell cycle progression in CGNPs, (ii) if G(1) regulation by Shh resembles that of classical mitogens, and (iii) whether individual D-type cyclins are essential components of Shh proliferative signaling in CGNPs. Our results indicate that Shh can drive continued cycling in immature, proliferating CGNPs. Shh treatment resulted in sustained activity of the G(1) cyclin-Rb axis by regulating levels of cyclinD1, cyclinD2, and cyclinE mRNA transcripts and proteins. Analysis of CGNPs from cyclinD1(-/-) or cyclinD2(-/-) mice demonstrates that the Shh proliferative pathway does not require unique functions of cyclinD1 or cyclinD2 and that D-type cyclins overlap functionally in this regard. In contrast to many known mitogenic pathways, we show that Shh proliferative signaling is mitogen-activated protein kinase independent. Furthermore, protein synthesis is required for early effects on cyclin gene expression. Together, our results suggest that Shh proliferative signaling promotes synthesis of regulatory factor intermediates that upregulate or maintain cyclin gene expression and activity of the G(1) cyclin-Rb axis in proliferating granule neuron precursors.

Rescue of alpha-SNS sodium channel expression in small dorsal root ganglion neurons after axotomy by nerve growth factor in vivo.

Small (18-25 microm diam) dorsal root ganglion (DRG) neurons are known to express high levels of tetrodotoxin-resistant (TTX-R) sodium current and the mRNA for the alpha-SNS sodium channel, which encodes a TTX-R channel when expressed in oocytes. These neurons also preferentially express the high affinity receptor for nerve growth factor (NGF), TrkA. Levels of TTX-R sodium current and of alpha-SNS mRNA are reduced in these cells after axotomy. To determine whether NGF participates in the regulation of TTX-R current and alpha-SNS mRNA in small DRG neurons in vivo, we axotomized small lumbar DRG neurons by sciatic nerve transection and administered NGF or Ringer solution to the proximal nerve stump using osmotic pumps. Ten to 12 days after pump implant, whole cell patch-clamp recording demonstrated that TTX-R current density was decreased in Ringer-treated axotomized neurons (154 +/- 45 pA/pF; mean +/- SE) compared with nonaxotomized control neurons (865 +/- 123 pA/pF) and was restored partially toward control levels in NGF-treated axotomized neurons (465 +/- 78 pA/pF). The V1/2 for steady-state activation and inactivation of TTX-R currents were similar in control, Ringer- and NGF-treated axotomized neurons. Reverse transcription polymerase chain reaction revealed an upregulation of alpha-SNS mRNA levels in NGF-treated compared with Ringer-treated axotomized DRG. In situ hybridization showed that alpha-SNS mRNA levels were decreased significantly in small Ringer-treated axotomized DRG neurons in vivo and also in small DRG neurons that were dissociated and maintained in vitro, so as to correspond to the patch-clamp conditions. NGF-treated axotomized neurons had a significant increase in alpha-SNS mRNA expression, compared with Ringer-treated axotomized cells. These results show that the administration of exogenous NGF in vivo, to the proximal nerve stump of the transected sciatic nerve, results in an upregulation of TTX-R sodium current and of alpha-SNS mRNA levels in small DRG neurons. Retrogradely transported NGF thus appears to participate in the control of excitability in these cells via actions that include the regulation of sodium channel gene expression in vivo.

Peripheral axotomy induces long-term c-Jun amino-terminal kinase-1 activation and activator protein-1 binding activity by c-Jun and junD in adult rat dorsal root ganglia In vivo.

One of the earliest documented molecular events after sciatic nerve injury in adult rats is the rapid, long-term upregulation of the immediate early gene transcription factor c-Jun mRNA and protein in lumbar dorsal root ganglion (DRG) neurons, suggesting that c-Jun may regulate genes that are important both in the early post-injury period and during later peripheral axonal regeneration. However, neither the mechanism through which c-Jun protein is increased nor the level of its post-injury transcriptional activity in axotomized DRGs has been characterized. To determine whether transcriptional activation of c-Jun occurs in response to nerve injury in vivo and is associated with axonal regeneration, we have assayed axotomized adult rat DRGs for evidence of jun kinase activation, c-Jun phosphorylation, and activator protein-1 (AP-1) binding. We report that sciatic nerve transection resulted in chronic activation of c-Jun amino-terminal kinase-1 (JNK) in L4/L5 DRGs concomitant with c-Jun amino-terminal phosphorylation in neurons, and lasting AP-1 binding activity, with both c-Jun and JunD participating in DNA binding complexes. The timing of JNK activation was dependent on the distance of the axotomy site from the DRGs, suggesting the requirement for a retrograde transport-mediated signal. AP-1 binding and c-Jun protein returned to basal levels in DRGs as peripheral regeneration was completed but remained elevated in the case of chronic sprouting, indicating that c-Jun may regulate target genes that are involved in axonal outgrowth.

Into reproductive health.

PIP: Now that the government of the Philippines' Department of Health (DOH) has endorsed the notion of reproductive health (RH), the current UNFPA-funded program, Strengthening the Management and Field Implementation of the Family Planning/Reproductive Health Program, has taken initial steps to implement the RH agenda of the 1994 International Conference on Population and Development. The program is being implemented by the DOH, LGU, and NGO tracks of the Philippine Family Planning Program, although the NGO track has progressed faster than the other two tracks in operationalizing RH. In light of its success, the NGO track will likely be used to develop and test models which may later be used in the other tracks. The authors present an overview of what has thus far been done to implement the reproductive health care agenda over the past 3 years under the NGO track. Reproductive tract infection (RTI) and HIV prevention, the detection and treatment of RTIs, syndromic case management, integrating RH into RTI/HIV services, preliminary results, human sexuality, maternal care, RH service statistics and management information systems, and lessons learned are discussed.^ieng

Timing of c-jun protein induction in lumbar dorsal root ganglia after sciatic nerve transection varies with lesion distance.

In situ hybridization and immunohistochemical studies have shown that sciatic nerve crush or transection induces upregulation of the immediate early gene c-jun mRNA and protein in lumbar dorsal root ganglion neurons. Here we have used enhanced chemiluminescent (ECL) immunoblotting as a sensitive and quantitative way of measuring the time of course of c-jun protein induction following sciatic nerve transection at two distances from the L4 and L5 dorsal root ganglia. c-Jun protein was first detected within 3 h of proximal sciatic nerve transection and within 6 h of distal nerve transection. These results indicate substantially earlier increases in c-jun protein after nerve injury than previously reported, which can be attributed to the sensitivity of this detection method. The earlier induction of c-jun after proximal as compared to distal nerve transection supports the hypothesis that the c-jun response to sciatic nerve injury involves a distance-dependent signalling mechanism.

Temporal variability of jun family transcription factor levels in peripherally or centrally transected adult rat dorsal root ganglia.

Enhanced chemiluminescent (ECL) immunoblotting was used to quantitatively assess the initial changes in jun family transcription factor protein levels in adult rat lumbar dorsal root ganglia (DRG) after peripheral axotomy and dorsal root transection, and to study the effects of neurotrophic factor administration on these changes. Transection of central (dorsal root) or peripheral (spinal nerve) branches of DRG neurons resulted in rapid elevation of c-jun protein levels, which was transient after dorsal root transection but sustained, though slightly attenuated, after spinal nerve transection. These results suggest that injury-induced c-jun elevation is biphasic, consisting of an early, transient, injury-initiated phase and a more prolonged secondary phase specific to peripheral target disconnection. c-jun protein changes were not modulated by administration of NGF or BDNF. Immunohistochemistry was used to localize c-jun protein induction to DRG neurons. Using ECL immunoblotting, we also observed temporally regulated increases in junD protein levels after both injuries. A transient up-regulation of junB was detected by immunoblotting 5 days after peripheral axotomy, coincident with a slight decrease in c-jun protein levels.

Sex education and AIDS education in the schools: what states and large school districts are doing.

Four-fifths of the states either require or encourage the teaching of sex education in the public schools, and nearly nine in 10 large school districts across the United States support such instruction. All but four states and virtually every large school district support the provision of AIDS education, and backing for instruction about sexually transmitted diseases and about abstinence is nearly as widespread. Fewer states and districts (two-thirds and four-fifths, respectively) require or encourage the schools to teach about pregnancy prevention. Examination of state and district curricula indicates that large school districts tend to be bolder than the states in the range of sex-related topics covered, especially in the area of pregnancy prevention. Districts also offer local educators more support (through curricula, training and other activities) than do the states. Finally, AIDS education appears to be receiving more attention and funding from both the states and the local school districts than is sex education.

Expanding Medicaid coverage for pregnant women: estimates of the impact and cost.

An estimated 361,000 pregnant women are expected to be newly eligible for Medicaid coverage when all states raise the income ceiling for such coverage to 100 percent of the federal poverty level by 1990, as Congress has mandated. According to a methodology for projecting the effects of recent congressional changes in the Medicaid program, about 64 percent of these women would be otherwise uninsured, at least for maternity care, and the rest would have some insurance, so Medicaid would be the payer of last resort. Congress has also given states the option to cover pregnant women with incomes from 100 to 185 percent of poverty. If all states were to do so, another 552,000 women would become eligible, 29 percent of whom would otherwise have no insurance coverage for maternity care. The estimate of newly eligible women with incomes below 185 percent of poverty represents 24 percent of the 3.8 million women who give birth in the United States each year. Under the 100-percent-of-poverty ceiling, the estimated number of poor women eligible for coverage ranges from 4,000 or fewer in 18 states and the District of Columbia to 41,000 in California and Texas. At 185 percent of poverty, the number ranges from 4,000 or fewer in 11 states and the District of Columbia to more than 90,000 in California and Texas. Eight states have already elected to extend Medicaid coverage to the 185-percent-of-poverty ceiling.(ABSTRACT TRUNCATED AT 250 WORDS)

Medicaid expenditures for maternity and newborn care in America.

An Alan Guttmacher Institute (AGI) survey of the Medicaid programs in each state and the District of Columbia found that some 542,000 low-income women have a Medicaid-subsidized delivery each year--about 15 percent of all women who give birth. The proportion ranges from three percent in Alaska to 25 percent in Michigan. The federal and state governments spend almost $1.2 billion annually for maternity care (including prenatal, postpartum and newborn care); the average expenditure per patient is $2,200. Tennessee reports the highest expenditure per patient ($3,500) and Louisiana the lowest ($1,300). Only the highest payments under Medicaid are close to charges for maternity care in the open market, a fact that results in a significant disincentive for physicians and hospitals to accept Medicaid patients. The $1.2 billion spent for Medicaid-subsidized maternity care compares with an estimated $11.5 billion spent for such care nationwide. Thus, Medicaid pays for about 10 percent of the nation's maternity care bill, although Medicaid subsidizes deliveries for 15 percent of all women who give birth. The figures for maternity care do not include Medicaid expenditures for neonatal intensive care, which, for the 17 states reporting data, average about $11,800 per infant. Although only about six percent of all newborns whose deliveries are subsidized by Medicaid require neonatal intensive care, such care is so expensive that it adds about 30 percent to all Medicaid expenditures for maternity care. Increased Medicaid payments for maternity care, including prenatal care, could have a positive impact on health outcomes for low-income mothers and their babies, and could reduce the necessity for massive and expensive medical treatment for newborns.

School-based clinics: a national conference.

PIP: On October 9-12, 1985, 250 health personnel, educators, and social service workers attended the 2nd national conference on school based clinics in the US. The conference, held in Chicago, was sponsored jointly by the Center for Population Options (CPO), a Washington based organization which provides technical assistance to groups interested in establishing school clinics, and the Ounce of Prevention Fund, a group of funding agencies in Illinois which works in cooperation with the state government to provide funds for school clinics in Illinois. The growth and accomplishments of the school based clinic movement in the US was reviewed in opening remarks made by the chairperson of the CPO. In 1984, at the time of the 1st national conference, there were clinics in only about 12 communities throughout the nation. Currently, there are clinics in about 50 communities located in 26 states. The clinics provide primary health care, including physical exams, immunizations, treatment for minor illnesses, counseling, nutrition assistance, gynecological exams, and family planning counseling. Some of the clinics dispense contraceptives. Most of the clinics do not provide abortion referrals. The clinics are generally operated by groups outside the educational system, e.g., hospitals, health departments, and nonprofit organizations. The schools furnish space for the clinics. Clinics are usually staffed by a nurse practitioner and a social worker with a backup physician. Topics discussed by the conference participants included strategies for establishing clinics and for gaining community and student acceptance, clinic evaluation, and funding issues. Controversy frequently accompanies the establishment of new clinics. Participants tended to agree that an essential element in launching a successful program is the establishment of a community advisory committee. A concerted effort must be made to address all community concerns about the clinic. Participants noted that it was best to obtain the support of school board members, administrators, and superintendents before approaching the staff at individual schools. Efforts must be made to gain the student's confidence in the ability of the clinics to serve their needs in a confidential manner. This goal can be more effectively met if the clinic is staffed by full-time rather than part-time workers. Some clinics successfully avoided initial controversy by deferring the provision of contraceptive services until their 2nd year of operation. Available but limited evaluation data suggest that clinic utilization is relatively high and that the clinics have had a negative impact on teenage pregnancy and a positive impact on contraceptive use among adolescents. For example, in St. Paul, Minnesota, a school clinic program was introduced in the 1970s, and between 1976-77 and 1984-85, the teenage birth rate declined from 59/1000 to 37/1000. The proportion of female students who used family planning increased from 7%-35% between 1976-77 and 1983-84. During the conference, the CPO announced plans to conduct a much needed large-scale evaluation of school clinics. The 3-year study will cover 7 geographical areas. The cost of operating the clinics is estimated to be US$100-US$125/student. Funding for the clinics comes primarily from the federal government sources including Maternal and Child Health and Social Services block grants, Medicaid, Title X of the Public Health Service Act, and the Adolescent Family Life Act. State governments and private donor organizations also support the clinics. A few clinics offset their expenses by charging minimal student fees. The participants tended to agree that in the long run, public funds should be used to support the programs. There was some disagreement among participants as to whether the primary task of the clinics was to improve the health status of adolescents or reduce the teenage pregnancy rate.^ieng

Paying for maternity care.

PIP: The costs of prenatal care and the delivery of newborns are continuously increasing. In the 3 years since 1982 alone, the cost of a hospital delivery has increased approximately 40%. 40% of all births in the US are to women aged 18-24. These women compose the highest risk group for having complications of pregnancy. It is alarming that in 1984 more than 25% of these women had no form of insurance to cover the costs. Poor, minority, and unemployed women are most likely to lack coverage. The 3 basic types of coverage are individual or direct, employer's or indirect, and federal. Direct insurance is not always affordable and often provides incomplete coverage. Employer's insurance is often able to cover the costs of maternity care for many young women. However, a high rate of job turnover and the loss of a husband due to death or divorce excludes teenagers, widows, and divorcees from maintaining this type of indirect insurance. Federal insurance in the form of Medicaid has strict eligibility requirements. In nearly 1/2 the states one must be below the poverty level in order to be eligible, and the benefits vary among the states. In addition, many practitioners will not accept Medicaid as payment. The Aid to Families With Dependent Children is available in lieu of Medicaid, but only to single mothers who already have dependent children. The Maternal Child Health block grant is designed to equalize the differences in Medicaid eligibility among states and to give coverage to poor women who are ineligible for Medicaid. The individual states are allowed to allot the monies for this grant without qualifications for minimum services, with the result that it is unknown which women receive necessary services.^ieng

Title X and its critics.

PIP: On Arpil 5, 1984 Senator Jesse Helms (R.--NC) was the leadd witness at a hearing before the US Senate Labor and Human Resources Subcommittee on Family and Human Services. The subcommittee was considering the renewal of Title X of the Public Health Service Act, the main national family planning legislation. Helm's prepared text (given in this article) was noteworthy not only for its hyperbole but also for its tone of intense frustration and for its repetition of the main themes enunciated by those less well known and less powerful who have opposed the program since it began. Title X was 1st enacted in 1970 and was reenacted in 1973, 1975, 1977, 1978 and 1981. The program has always had its share of critics; but in the late 1970's with the emergence of the New Right, such groups moved into political power. Title X provided for project grants to be made to public and private nonprofit agencies to provide contraceptive services along with training, technical assistance and other support activities. It also spelled out government responsibilities and functions in relation to human reproduction and population research; and it created the post of Deputy Assistant Secretary for Population Affairs, with direct responsibility for both types of activities within the Department of Health and Human Services. Eligibility for services was broad--applying to all who need and want services, with priority for low-income persons. A requirement of voluntary participation and a prohibition of the use of Title X funds for abortion as a family planning method were clearly stated. Title X has been criticized by Catholics and many other groups. This article attempts to meet criticisms aimed at the functioning, appropriateness and impact of the Title X program, and summarizes the findings of a large body of socialogical and demographic research that has direct or indirect bearing on the issues raised by the critics.^ieng